Bisphosphonate quinolone conjugates and uses thereof

ABSTRACT

Described herein are bisphosphonate quinolone compounds, conjugates and pharmaceutical formulations thereof that can include a bisphosphonate and a quinolone, where the quinolone can be releasably coupled to the bisphosphonate. Also provided herein are methods of making and methods of using the bisphosphonate quinolone compounds, conjugates and pharmaceutical formulations thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US2017/035764, filedJun. 2, 2017, that claims the benefit of and priority to: U.S.Provisional Patent Application No. 62/345,370, filed on Jun. 3, 2016,entitled “BONE TARGETED THERAPEUTICS AND DIAGNOSTICS;” U.S. ProvisionalPatent Application No. 62/357,727, filed on Jul. 1, 2016, entitled“BISPHOSPHONATE QUINOLONE BIOCONJUGATES AND USES THEREOF;” and U.S.Provisional Patent Application No. 62/448,060, filed on Jan. 19, 2017,entitled “BISPHOSPHONATE QUINOLONE BIOCONJUGATES AND USES THEREOF,” thecontents of all of which are incorporated by reference herein in theirentirety.

This application also claims the benefit of and priority to co-pendingU.S. Provisional Patent Application No. 62/695,583, filed Jul. 9, 2018,entitled “BISPHOSPHONATE QUINOLONE CONJUGATES AND USES THEREOF,” thecontents of which are incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number1R41DE025789-01 awarded by the NIH/NIDCR. The government has certainrights in the invention.

BACKGROUND

Infectious bone disease, also referred to as osteomyelitis, jawboneinfections, and other bone infections, is a significant problem in humanand animal health and can have devastating consequences from limb lossto fatality. Due to the inherent difficulties bone presents, treatmentof osteomyelitis and other bone infections is typically long anddifficult and often requires surgical intervention. Therefore, thereexists a long-felt and unmet need for improved treatments forosteomyelitis in all its forms or clinical subtypes and other boneinfections.

SUMMARY

Provided herein, in some aspects, are BP quinolone compounds andconjugates that can contain a bisphosphonate (BP) that can be releasablyconjugated to a quinolone, such as ciprofloxacin or moxifloxacin. Inembodiments, the BP quinolone conjugate can selectively deliver aquinolone to bone, bone grafts, and or bone graft substitutes (i.e. cantarget bone, bone grafts, or bone graft substitutes) in a subject. Insome embodiments, the BP quinolone conjugate can release the quinolone.Also provided herein are methods of synthesizing BP quinolone conjugatesand methods of killing or inhibiting bacteria growth and of treating orpreventing bone diseases with abnormal bone resorption, osteoporosis,osteomyelitis, osteonecrosis, peri-implantitis, periodontis, and/orother bone infections with one or more of the BP quinolone compounds andconjugates provided herein.

In some aspects the conjugate can be a compound according to Formula (6)

Also provided herein are pharmaceutical compositions containing acompound according to Formula (6) and a pharmaceutically acceptablecarrier.

Also provided herein are methods of treating a bone infection in asubject in need thereof that can include the step of administering anamount of the compound according to Formula (6) or a pharmaceuticalformulation containing a compound according to Formula (6) to a subjectin need thereof.

Also provided herein are compounds and conjugates containing abisphosphonate (BP) and a quinolone compound, wherein the quinolonecompound is releasably coupled to the bisphosphonate via a linker. TheBP can be selected from the group of: hydroxyl phenyl alkyl or arylbisphosphonates, hydroxyl phenyl (or aryl) alkyl hydroxylbisphosphonates, amino phenyl(or aryl) alkyl bisphosphonates, aminophenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkylbisphosphonates, hydroxyl alkyl hydroxyl bisphosphonates, hydroxyl alkylphenyl(or aryl) alkyl bisphosphonates, hydroxyl phenyl(or aryl) alkylhydroxyl bisphosphonates, amino phenyl(or aryl) alkyl bisphosphonates,amino phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkylbisphosphonates, hydroxyl alkyl hydroxyl bisphosphonates, hydroxypyridylalkyl bisphosphonates, pyridyl alkyl bisphosphonates, hydroxyl imadazoylalkyl bisphosphonates, imidazoyl alkyl bisphosphonates, etidronate,pamidronate, neridronate, olpadronate, alendronate, ibandronate,risedronate, zoledronate, minodronate and combinations thereof, whereinall the compounds can be optionally further substituted or areunsubstituted. The quinolone compound can be a fluoroquinolone. Thequinolone compound can be selected from the group of: alatrofloxacin,amifloxacin, balofloxacin, besifloxacin, cadazolid, ciprofloxacin,clinafloxacin, danofloxacin, delafloxacin, difloxacin, enoxacin,enrofloxacin, finafloxacin, flerofloxacin, flumequine, gatifloxacin,gemifloxacin, grepafloxacin, ibafloxacin, JNJ-Q2, levofloxacin,lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin,ofloxacin, orbifloxacin, pazufloxacin, pefloxacin, pradofloxacin,prulifloxacin, rufloxacin, sarafloxacin, sitafloxacin, sparfloxacin,temafloxacin, tosufloxacin, trvafloxacin, zabofloxacin, nemonoxacin andcombinations thereof.

The quinolone compound can have a structure according to Formula A,

where R¹ can be substituents including alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups,

where R² can be substituents including alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups,

where R³ can be substituents including alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups, and

where R⁴ can be substituents including alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups.

In any one or more aspects, the linker can be a carbamate linker. Thelinker can be an aryl carbamate linker. The linker can be an O-thioarylcarbamate linker. The linker can be an S-thioaryl carbamate linker. Thelinker can be a phenyl carbamate linker (either substituted orunsubstituted). The linker can be a thiocarbamate linker. The linker canbe a O-thiocarbamate linker. The linker can be an S-thiocarbamatelinker. The linker can be an O-carbamate linker. The linker can be anactivated carbamate, for example a phosphonyl carbamate such as inFormula (41) and Formula (43) herein. The activated carbamate can be anaryl or a phosphonyl substituted carbamate. The linker can be attachedto the R¹ group of Formula A.

In any one or more aspects, the alpha position of theethylidenebisphosphonate can be substituted by hydroxy, fluoro, chloro,bromo or iodo. In some aspects, the bisphosphonate can include apara-hydroxyphenylethylidene group or derivative thereof. In someaspects, ethylidenebisphosphonate does not contain an alpha-hydroxy atthe alpha position.

In some aspects, the compound has a formula according to Formula (12):

In some aspects, the compound has a formula according to Formula (13),

In some aspects, the compound has a formula according to Formula (15),

In some aspects, the compound has a formula according to Formula (41) orFormula (43),

Also provided herein are pharmaceutical formulations that can contain abisphosphonate and a quinolone compound, wherein the quinolone compoundis releasably coupled to the bisphosphonate via a linker; and apharmaceutically acceptable carrier. The bisphosphonate can be selectedfrom the group of: hydroxyl phenyl alkyl or aryl bisphosphonates,hydroxyl phenyl (or aryl) alkyl hydroxyl bisphosphonates, aminophenyl(or aryl) alkyl bisphosphonates, amino phenyl(or aryl) alkylhydroxyl bisphosphonates, hydroxyl alkyl bisphosphonates, hydroxyl alkylhydroxyl bisphosphonates, hydroxyl alkyl phenyl(or aryl) alkylbisphosphonates, hydroxyl phenyl(or aryl) alkyl hydroxylbisphosphonates, amino phenyl(or aryl) alkyl bisphosphonates, aminophenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkylbisphosphonates, hydroxyl alkyl hydroxyl bisphosphonates, hydroxypyridylalkyl bisphosphonates, pyridyl alkyl bisphosphonates, hydroxyl imadazoylalkyl bisphosphonates, imidazoyl alkyl bisphosphonates, etidronate,pamidronate, neridronate, olpadronate, alendronate, ibandronate,risedronate, zoledronate, minodronate, methylenehydroxy bisphosphonate,ethylidene bisphosphonate, and combinations thereof, wherein all thecompounds can be optionally further substituted or are unsubstituted.

The quinolone compound can be a fluoroquinolone. The quinolone compoundcan be selected from the group of: alatrofloxacin, amifloxacin,balofloxacin, besifloxacin, cadazolid, ciprofloxacin, clinafloxacin,danofloxacin, delafloxacin, difloxacin, enoxacin, enrofloxacin,finafloxacin, flerofloxacin, flumequine, gatifloxacin, gemifloxacin,grepafloxacin, ibafloxacin, JNJ-Q2, levofloxacin, lomefloxacin,marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, ofloxacin,orbifloxacin, pazufloxacin, pefloxacin, pradofloxacin, prulifloxacin,rufloxacin, sarafloxacin, sitafloxacin, sparfloxacin, temafloxacin,tosufloxacin, trvafloxacin, zabofloxacin, nemonoxacin and combinationsthereof.

In some aspects, the BP is etidronate. In some aspects, the quinolone isciprofloxacin or moxifloxacin. In other aspects, the BP can be anotherBP described herein, such as pamidronate, neridronate, olpadronate,alendronate, ibandronate, minodronate, risedronate, zoledronate,hydroxymethylenebisphosphonate, and combinations thereof. In someaspects, the quinolone is ciprofloxacin or moxifloxacin.

The quinolone compound can have a structure according to Formula A,

where R¹ can be substituents including alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups,

where R² can be substituents including alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups,

where R³ can be substituents including alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups, and

where R⁴ can be substituents including alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups.

In any one or more aspects, the linker can be a carbamate linker. Thelinker can be an aryl carbamate linker. The linker can be an O-thioarylcarbamate linker. The linker can be an S-thioaryl carbamate linker. Thelinker can be a phenyl carbamate linker (either substituted orunsubstituted). The linker can be a thiocarbamate linker. The linker iscan be a O-thiocarbamate linker. The linker can be an S-thiocarbamatelinker. The linker can be an O-carbamate linker. The linker can be anactivated carbamate, for example a phosphonyl carbamate such as inFormula (41) and Formula (43) herein. The activated carbamate can be anaryl or a phosphonyl substituted carbamate. The linker can be attachedto the R¹ group of Formula A.

In some aspects, the alpha position of the ethylidenebisphosphonate canbe substituted by hydroxy, fluoro, chloro, bromo or iodo. In someaspects, the bisphosphonate can include a para-hydroxyphenylethylidenegroup or derivative thereof. In some aspects, ethylidenebisphosphonatedoes not contain an alpha-hydroxy at the alpha position.

In some aspects, the compound has a formula according to Formula (12):

In some aspects, the compound has a formula according to Formula (13),

In some aspects, the compound has a formula according to Formula (15),

In some aspects, the compound has a formula according to Formula (41) orFormula (43),

In various aspects, a compound or a conjugate is provided thatcomprises: a bisphosphonate (BP); and a quinolone compound; wherein thequinolone compound is releasably coupled to the bisphosphonate via alinker. The linker can be a carbamate linker, as described herein. Thelinker can be an aryl carbamate, an aryl thiocarbamate, an O-thioarylcarbamate, an S-thioaryl carbamate, a thiocarbamate (such as anO-thiocarbamate or an S-thiocarbamate), a phenyl carbamate (eithersubstituted or unsubstituted), an O-carbamate, or a phosphonyl carbamate(such as in Formula (41) and Formula (43) herein). The linker can be anactivated carbamate, such as an aryl or a phosphonyl substitutedcarbamate. The bisphosphonate, quinolone compound and linker can be anyof those provided herein. In an aspect, the compound can comprise abisphosphonate (BP), quinolone and a linker, wherein the BP is analpha-OH containing BP and the quinolone is indirectly conjugated to theBP at the geminal end of the P by the linker. A pharmaceuticalcomposition is also provided comprising an amount of the compound orconjugate as set forth in any one or more of the aspects providedherein, and a pharmaceutically acceptable carrier.

In various aspects, a method is provided comprising: administering anamount of the compound or conjugate as set forth in any one or moreaspects provided herein, or a pharmaceutical formulation thereof, to asubject.

The amount of the compound in the pharmaceutical formulation can be anamount effective to kill or inhibit bacteria growth. The amount of thecompound in the pharmaceutical formulation can be an amount effective totreat or prevent bone diseases with abnormal bone resorption,osteoporosis, osteomyelitis, osteonecrosis, peri-implantitis, and/orperiodontitis.

Also provided herein are methods of treating or preventing osteomyelitisin a subject in need thereof that can include the step of administeringan amount of a compound as provided herein or pharmaceutical formulationthereof to the subject in need thereof.

Also provided herein are methods of treating or preventingperi-implantitis or periodontitis in a subject in need thereof, themethod comprising administering an amount of administering an amount ofa compound as provided herein or pharmaceutical formulation thereof tothe subject in need thereof.

Also provided herein are methods of treating or preventing diabetic footin a subject in need thereof, the method comprising administering anamount of administering an amount of a compound as provided herein orpharmaceutical formulation thereof to the subject in need thereof.

Also provided herein are bone graft compositions that can include a bonegraft material and a compound as described herein or a pharmaceuticalformulation thereof, wherein the compound or pharmaceutical formulationthereof is attached to, integrated with, chemisorbed to, or mixed withthe bone graft material. The bone graft material can be autograft bonematerial, allograft bone material, xenograft bone material, a syntheticbone graft material, or any combination thereof.

Also provided herein are methods that can include the step of implantingthe bone graft composition as described herein in a subject in needthereof.

Also provided herein are methods of treating or preventing biofilminfection at an osseous or implant surgical site, or at a surgical sitewhere bone grafting is performed, where the methods can include the stepof administering a compound as described herein to a subject in needthereof.

Also provided herein are methods of treating or preventing biofilminfection at an osseous or implant surgical site, or at a surgical sitewhere bone grafting is performed, where the method can include the stepof implanting a bone graft composition as described herein to a subjectin need thereof.

Other compounds, compositions, formulations, methods, features, andadvantages of the present disclosure of a fabrication system fornanowire template synthesis, will be or become apparent to one withskill in the art upon examination of the following drawings and detaileddescription. It is intended that all such additional systems, methods,features, and advantages be included within this description, be withinthe scope of the present disclosure, and be protected by theaccompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciatedupon review of the detailed description of its various embodiments,described below, when taken in conjunction with the accompanyingdrawings.

FIG. 1 shows a scanning electron micrograph (SEM; 100× magnification) ofa surgical specimen from a patient with chronic osteomyelitis showingcharacteristic multi-layered and matrix-enclosed biofilms colonizingbone surfaces internally and externally; inset top right showshigh-power view (5000× magnification) of the causative staphylococcalbiofilm pathogens. [The sample was processed for SEM, sputter coatedwith platinum and imaged with an XL 30S SEM (FEG, FEI Co., Hillsboro,Oreg.) operating at 5 kV in the secondary electron mode].

FIGS. 2A-2B shows general synthesis schemes of a phenyl carbamateBP-ciprofloxacin conjugate.

FIG. 3 shows a table demonstrating the AST and MIC results forciprofloxacin and BP-ciprofloxacin against a panel of clinical S. aureusosteomyelitis pathogens.

FIG. 4 shows a graph demonstrating the results from a spectroscopicanalysis of BP-ciprofloxacin conjugate in trypticase soy brothmicrobiological media at 0 hr and at 24 hrs for the variousconcentrations of the conjugate used in antimicrobial susceptibilitytesting in vitro; no degradation is observed after 24 hrs, which is thetypical length of an experimental period for in vitro antimicrobialtesting, indicating excellent stability of the antimicrobial. [*resultsfor 0.24-3.9 mcg/mL (red bars) are inconclusive because of a high valueof “blank” measurements]

FIG. 5 shows a graph demonstrating the results of a spectroscopicanalysis of one BP-ciprofloxacin conjugate (BP-carbamate-Ciprofloxacin,BCC, compound 6) in trypticase soy broth microbiological media with theaddition of HA spherules; the significant decreases from 0 hr to 24 hrsconfirms conjugate adsorption to HA since only the supernatant ismeasured absent the HA spherules with adsorbed conjugate. [results for1.95-250 mcg/mL are all statistically significant: p<0.05, ANOVA;triplicate; *results for 0.12-0.48 mcg/mL (red bars) are inconclusivebecause of a high value of “blank” measurements].

FIG. 6 shows graphs demonstrating the results from antimicrobialsusceptibility testing of BP-ciprofloxacin against planktonic culturesof S. aureus strain ATCC-6538 shows an improved bactericidal profile inacidic (right graph) versus basic (left graph) pH.

FIG. 7 shows graphs demonstrating the time-kill results forBP-ciprofloxacin (conjugate) against S. aureus strain ATCC-6538 (rightgraph) and MRSA strain MR4-CIPS (left graph) and at 1× (red line) and ½×(black line) the established MICs showing strong bactericidal activityat 1 hr and up to 24 hrs.

FIG. 8 shows graphs demonstrating results from antimicrobialsusceptibility testing of BP-ciprofloxacin against biofilms of S. aureusstrain ATCC-6538 (top graph) and P. aeruginosa strain ATCC-15442 (bottomgraph) formed on polystyrene as a substrate.

FIG. 9 shows graphs demonstrating results from antimicrobialsusceptibility testing of BP-ciprofloxacin against biofilms of S. aureusstrain ATCC-6538 (left graph) and P. aeruginosa strain ATCC-15442 (rightgraph) formed on HA discs as the substrate. All tested concentrations ofthe conjugate (orange bars) resulted in statistically significantbactericidal activity against S. aureus including ciprofloxacin alone(red bar). [*p<0.05, Kruskal-Wallis test; triplicate].

FIG. 10 shows a graph demonstrating results from preventativeexperiments where HA spherules are pre-coated with BP-ciprofloxacin andthen inoculated with S. aureus. Control (C: red bar) represents culturedbacteria without HA and not treated with conjugate, and after 24 hrs anexpected significant increase in planktonic growth is observed when thesupernatant is measured. Control+HA (C+HA bar) represents culturedbacteria with HA, but still no treatment, and after 24 hrs somebacterial growth is observed but not as much as the HA negative control(red bar) because bacteria bind to HA and form biofilms which are notmeasured in the HA free supernatant. Comparing these controls to thetreatments we can see that at 7.8 to 250 mcg/mL of the conjugate thereis complete bacterial inhibition after 24 h. At lower concentrationsranging from 0.12 to 3.9 mcg/mL bacteria grew slightly but were stillstrongly inhibited.

FIG. 11 shows a table demonstrating the survival of biofilm bacteriaafter 24 hr incubation in presence of BP-ciprofloxacin coated HA discs.

FIG. 12 shows a graph demonstrating the antimicrobial results from invivo animal testing showing efficacy of tested compounds for reducingbacterial load. The conjugate showed the greatest efficacy at 0.9 mg/kgtotal given in multiple doses, with no recoverable bacteria. Next asingle dose of 10 mg/kg of the conjugate demonstrated 2 log reduction(99% bactericidal activity) as compared to the negative control, andnearly 1 log greater bactericidal activity as compared to the multipledosing regimen of ciprofloxacin alone which demonstrated a 1 logreduction.

FIG. 13 demonstrates the general BP quinolone conjugate targetingstrategy.

FIG. 14 demonstrates a general strategy of a BP quinolone conjugatecapable of targeting and releasing.

FIG. 15 shows an embodiment of a BP-FQ conjugate.

FIG. 16 shows a synthesis scheme for a BP-FQ conjugate.

FIG. 17 shows antimicrobial susceptibility testing results forciprofloxacin, BCC (compound 6) and BP-Amide-Ciprofloxacin (BAC,compound 11) tested against a panel of clinically relevant S. aureusosteomyelitis pathogens. (MSSA=methicillin-susceptible S. aureus;MRSA=methicillin-resistant S. aureus).

FIG. 18 shows a graph demonstrating results of a spectroscopic analysisof BCC (compound 6) in microbiological media with the addition of HAmicrospherules confirms adsorption of conjugate to HA, as evidenced bythe significant decreases from 0 hr to 24 hrs since only the supernatantis measured absent the HA spherules with adsorbed conjugate. [resultsfor 1.95-250 mcg/mL are all statistically significant: p<0.05, ANOVA;triplicate; *results for 0.12-0.48 mcg/mL (red bars) are inconclusivebecause of a high value of “blank” measurements.

FIG. 19 shows a graph demonstrating efficacy of the BCC (compound 6) forreducing bacterial load or mean CFU/gram of tissue. The greatestefficacy was again observed at a single high dose (10 mg/kg) of theconjugate as compared to the control [*p=0.0005; unpaired t-test, errorbars represent Standard Error].

FIG. 20 shows additional BP-Ab conjugate design.

FIG. 21 shows an embodiment of a synthesis scheme for synthesis of BP-Abconjugates with an O-thiocarbamate linker.

FIG. 22 shows an embodiment of a scheme for synthesis of Q-OH protectedBP esters.

FIG. 23 shows an embodiment of a scheme for synthesis of BP 3-linker3-ciprofloxacin.

FIG. 24 shows a graph and image demonstrating results from an evaluationof the MIC of an O-thiocarbamate BP conjugate against planktonic S.aureus strain ATCC 6538: negative control=medium+microbes withoutconjugate treatment; positive control=sterile medium without microbes.

FIG. 25 shows a graph demonstrating results from an evaluation of theantimicrobial activity or bacterial load reduction of the thiocarbamateconjugate against biofilms of S. aureus strain ATCC 6538 formed onpolystyrene as the substrate: negative control=microbial dilutionwithout conjugate treatment; positive control=sterile dilution withoutmicrobes.

FIG. 26 shows a graph demonstrating results from an evaluation of theantimicrobial activity of the O-thiocarbamate BP conjugate testedagainst preformed biofilms of S. aureus ATCC 6538 on hydroxyapatite asthe substrate; negative control=microbial dilution without conjugatetreatment. (*p<0.05, Kruskal-Wallis test; triplicate;comparator=control).

FIG. 27 shows a graph demonstrating results from a study usingO-thiocarbamate BP conjugate-treated hydroxyapatite discs evaluating theability to prevent biofilm formation of S. aureus ATCC 6538; negativecontrol=microbial dilution without conjugate treatment. (*p<0.05,Kruskal-Wallis test; triplicate; comparator=control).

FIG. 28 shows a graph demonstrating results from a study usingO-thiocarbamate BP conjugate-treated hydroxyapatite powder evaluatingthe ability to prevent biofilm formation of S. aureus ATCC 6538;negative control=microbial dilution without conjugate treatment.(*p<0.05, Kruskal-Wallis test; triplicate; comparator=control).

FIG. 29 shows an alpha-hydroxy modified risedronate and zoledronate.

FIG. 30 shows 1) a BP modified by substituting or removing thealpha-hydroxy group (p-PyrEBP); 2) a BP modified by substituting at thepara-position of pyridine ring (p-RIS). The circled H is attached to thealpha carbon of the bisphosphonate substituted carbon chain.

FIG. 31 shows a synthesis scheme for a BP-ciprofloxacin conjugate havingan amide linkage (BAC, compound 11) as opposed to a carbamate linkage.

FIG. 32 shows a graph demonst the results of a minimal inhibitoryconcentration (MIC) assay for 6 and 11 against eight S. aureus strainsusing microdilution methodology.

FIG. 33 shows graphs demonstrating antimicrobial susceptibility testingof 6 against biofilms of S. aureus strain ATCC-6538 (top graph) and P.aeruginosa strain ATCC-15442 (bottom graph) formed on HA discs as thesubstrate. All tested concentrations of 6 (dotted bars top graph) andthe parent antibiotic ciprofloxacin resulted in statisticallysignificant bactericidal activity against S. aureus; c=negative controlcomparator. Against P. aeruginosa, 6 was most effective at physiologicalpH at 8 μg/mL, and also effective at acidic pH at this concentration,but ciprofloxacin was inactive under either acidic or physiologicalconditions compared to the controls [*p<0.05, Kruskal-Wallis test;triplicate].

FIG. 34 shows graphs demonstrating the results from Antimicrobialsusceptibility testing (top graph) of 11 at increasing concentrationsagainst biofilms of S. aureus strain ATCC-6538 formed on HA as thesubstrate. No significant activity is observed at any concentration ascompared to the control C+[p>0.05, Kruskal-Wallis test; triplicate]. Thebottom graph shows results from preventative experiments where HA ispretreated with 11 or the parent antibiotic ciprofloxacin and theninoculated with S. aureus, and again no antimicrobial activity isobserved for 11; the only significant reduction is seen with the parentdrug at a relatively high dose of 400 μg/mL [*p<0.05, Kruskal-Wallistest; triplicate].

FIG. 35 shows a graph demonstrating antimicrobial susceptibility of 6against biofilms of Aggregatibacter actinomycetemcomitans strain D7S-5grown on HA shows an effective antimicrobial profile for conjugate 6at >15 μg/mL.

FIG. 36 shows a graph demonstrating antimicrobial results from in vivoanimal testing. Data show efficacy of tested compounds for reducingbacterial load. The greatest efficacy was observed at a single high dose(10 mg/kg) of 6 where a 2 log reduction (99% bactericidal activity) wasseen as compared to the negative control.

FIG. 37 shows a graph demonstrating Antimicrobial results from thesecond animal experiment. Data shows efficacy of 6 for reducingbacterial load or mean CFU/gram of tissue (y-axis). The greatestefficacy was observed at a single high dose (10 mg/kg) of the conjugatecompared to the control and the multiple low dose group (0.3 mg/kg×3)[*p=0.0005; unpaired t-test, errors bars represent Standard Error].

FIG. 38 shows a BP-carbamate-moxifloxacin BP conjugate and synthesisscheme.

FIG. 39 shows a BP-carbamate-gatifloxacin BP conjugate and synthesisscheme.

FIG. 40 shows a BP-p-Hydroxyphenyl Acetic Acid-ciprofloxacin BPconjugate and synthesis scheme.

FIG. 41 shows a BP-OH-ciprofloxacin BP conjugate and synthesis scheme.

FIG. 42 shows a BP-O-Thiocarbamate-ciprofloxacin BP conjugate andsynthesis scheme.

FIG. 43 shows a BP-S-Thiocarbamate-ciprofloxacin BP conjugate andsynthesis scheme.

FIG. 44 shows a BP-Resorcinol-ciprofloxacin BP conjugate and synthesisscheme.

FIG. 45 shows a BP-Hydroquinone-ciprofloxacin BP conjugate and synthesisscheme.

FIG. 46 shows one embodiment of a genus structure for a genus ofBP-Fluoroquinolones.

FIG. 47 shows various BP-fluoroquinolone conjugates.

FIG. 48 shows one embodiment of a genus structure for a genus of aphosphonate containing an aryl group.

FIG. 49 shows various BPs, where X can be F, Cl, Br, or I.

FIG. 50 shows various BP's with terminal primary amines.

FIG. 51 shows various BPs coupled to a linker containing a terminalhydroxyl and amine functional groups where R can be Risedronate,Zoledronate, Minodronate, Pamidronate, or Alendronate.

FIG. 52 shows various BP-pamidronate-ciprofloxacin conjugates.

FIG. 53 shows various BP-Alendronate-ciprofloxacin conjugates.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of molecular biology, microbiology,nanotechnology, pharmacology, organic chemistry, biochemistry, botanyand the like, which are within the skill of the art. Such techniques areexplained fully in the literature.

Definitions

Unless otherwise specified herein, the following definitions areprovided.

As used herein, “about,” “approximately,” and the like, when used inconnection with a numerical variable, generally refers to the value ofthe variable and to all values of the variable that are within theexperimental error (e.g., within the 95% confidence interval for themean) or within ±10% of the indicated value, whichever is greater.

As used interchangeably herein, “subject,” “individual,” or “patient,”refers to a vertebrate, preferably a mammal, more preferably a human.Mammals include, but are not limited to, murines, simians, humans, farmanimals, sport animals, and pets. The term “pet” includes a dog, cat,guinea pig, mouse, rat, rabbit, ferret, and the like. The term “farmanimal” includes a horse, sheep, goat, chicken, pig, cow, donkey, llama,alpaca, turkey, and the like.

As used herein, “control” can refer to an alternative subject or sampleused in an experiment for comparison purposes and included to minimizeor distinguish the effect of variables other than an independentvariable.

As used herein, “analogue,” such as an analogue of a bisphosphonatedescribed herein, can refer to a structurally close member of the parentmolecule or an appended parent molecule such as a bisphosphonate.

As used herein, “conjugated” can refer to direct attachment of two ormore compounds to one another via one or more covalent or non-covalentbonds. The term “conjugated” as used herein can also refer to indirectattachment of two or more compounds to one another through anintermediate compound, such as a linker.

As used herein, “pharmaceutical formulation” refers to the combinationof an active agent, compound, or ingredient with a pharmaceuticallyacceptable carrier or excipient, making the composition suitable fordiagnostic, therapeutic, or preventive use in vitro, in vivo, or exvivo.

As used herein, “pharmaceutically acceptable carrier or excipient”refers to a carrier or excipient that is useful in preparing apharmaceutical formulation that is generally safe, non-toxic, and isneither biologically or otherwise undesirable, and includes a carrier orexcipient that is acceptable for veterinary use as well as humanpharmaceutical use. A “pharmaceutically acceptable carrier or excipient”as used in the specification and claims includes both one and more thanone such carrier or excipient.

As used herein, “pharmaceutically acceptable salt” refers to any acid orbase addition salt whose counter-ions are non-toxic to the subject towhich they are administered in pharmaceutical doses of the salts.

As used herein, “active agent” or “active ingredient” refers to acomponent or components of a composition to which the whole or part ofthe effect of the composition is attributed.

As used herein, “dose,” “unit dose,” or “dosage” refers to physicallydiscrete units suitable for use in a subject, each unit containing apredetermined quantity of a BP conjugate, such as a BP quinoloneconjugate, composition or formulation described herein calculated toproduce the desired response or responses in association with itsadministration.

As used herein, “derivative” refers to any compound having the same or asimilar core structure to the compound but having at least onestructural difference, including substituting, deleting, and/or addingone or more atoms or functional groups. The term “derivative” does notmean that the derivative is synthesized from the parent compound eitheras a starting material or intermediate, although this may be the case.The term “derivative” can include prodrugs, or metabolites of the parentcompound. Derivatives include compounds in which free amino groups inthe parent compound have been derivatized to form amine hydrochlorides,p-toluene sulfonamides, benzoxycarboamides, t-butyloxycarboamides,thiourethane-type derivatives, trifluoroacetylamides,chloroacetylamides, or formamides. Derivatives include compounds inwhich carboxyl groups in the parent compound have been derivatized toform methyl and ethyl esters, or other types of esters, amides,hydroxamic acids, or hydrazides. Derivatives include compounds in whichhydroxyl groups in the parent compound have been derivatized to formO-acyl, O-carbamoyl, or O-alkyl derivatives. Derivatives includecompounds in which a hydrogen bond donating group in the parent compoundis replaced with another hydrogen bond donating group such as OH, NH, orSH. Derivatives include replacing a hydrogen bond acceptor group in theparent compound with another hydrogen bond acceptor group such asesters, ethers, ketones, carbonates, tertiary amines, imine, thiones,sulfones, tertiary amides, and sulfides. “Derivatives” also includesextensions of the replacement of the cyclopentane ring, as an example,with saturated or unsaturated cyclohexane or other more complex, e.g.,nitrogen-containing rings, and extensions of these rings with variousgroups.

As used herein, “administering” refers to an administration that isoral, topical, intravenous, subcutaneous, transcutaneous, transdermal,intramuscular, intra-joint, parenteral, intra-arteriole, intradermal,intraventricular, intracranial, intraperitoneal, intralesional,intranasal, rectal, vaginal, by inhalation, or via an implantedreservoir. The term “parenteral” includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, and intracranial injections orinfusion techniques.

The term “substituted” as used herein, refers to all permissiblesubstituents of the compounds described herein. In the broadest sense,the permissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,but are not limited to, halogens, hydroxyl groups, or any other organicgroupings containing any number of carbon atoms, e.g. 1-14 carbon atoms,and optionally include one or more heteroatoms such as oxygen, sulfur,or nitrogen grouping in linear, branched, or cyclic structural formats.

Representative substituents include alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl,carboxyl, substituted carboxyl, amino, substituted amino, amido,substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups.

As used herein, “suitable substituent” means a chemically andpharmaceutically acceptable group, i.e., a moiety that does notsignificantly interfere with the preparation of or negate the efficacyof the inventive compounds. Such suitable substituents may be routinelychosen by those skilled in the art. Suitable substituents include butare not limited to the following: a halo, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₂-C₆ alkynyl, C₃-C₈cycloalkenyl, (C₃-C₈ cycloalkyl)C₁-C₆ alkyl, (C₃-C₈ cycloalkyl)C₂-C₆alkenyl, (C₃-C₈ cycloalkyl)C₁-C₆ alkoxy, C₃-C₇ heterocycloalkyl, (C₃-C₇heterocycloalkyl)C₁-C₆ alkyl, (C₃-C₇ heterocycloalkyl) C₂-C₆ alkenyl,(C₃-C₇ heterocycloalkyl)C₁-C₆ alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C₁-C₆ alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C₁-C₆alkylamino, di-(C₁-C₆ alkyl)amino, carbamoyl, (C₁-C₆ alkyl)carbonyl,(C₁-C₆ alkoxy)carbonyl, (C₁-C₆ alkyl)aminocarbonyl, di-(C₁-C₆alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C₁-C₆alkyl)sulfonyl, and arylsulfonyl. The groups listed above as suitablesubstituents are as defined hereinafter except that a suitablesubstituent may not be further optionally substituted.

The term “alkyl” refers to the radical of saturated aliphatic groups(i.e., an alkane with one hydrogen atom removed), includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups.

In some embodiments, a straight chain or branched chain alkyl can have30 or fewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straightchains, and C₃-C₃₀ for branched chains). In other embodiments, astraight chain or branched chain alkyl can contain 20 or fewer, 15 orfewer, or 10 or fewer carbon atoms in its backbone. Likewise, in someembodiments cycloalkyls can have 3-10 carbon atoms in their ringstructure. In some of these embodiments, the cycloalkyl can have 5, 6,or 7 carbons in the ring structure.

The term “alkyl” (or “lower alkyl”) as used herein is intended toinclude both “unsubstituted alkyls” and “substituted alkyls,” the latterof which refers to alkyl moieties having one or more substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents include, but are not limited to, halogen, hydroxyl,carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl),thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido,amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, oran aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons in its backbone structure. Likewise, “lower alkenyl” and“lower alkynyl” have similar chain lengths.

It will be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude halogen, hydroxy, nitro, thiols, amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Cycloalkyls can besubstituted in the same manner.

The term “heteroalkyl,” as used herein, refers to straight or branchedchain, or cyclic carbon-containing radicals, or combinations thereof,containing at least one heteroatom.

Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se,B, and S, wherein the phosphorous and sulfur atoms are optionallyoxidized, and the nitrogen heteroatom is optionally quaternized.Heteroalkyls can be substituted as defined above for alkyl groups.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S— alkyl, —S-alkenyl, and—S-alkynyl. Representative alkylthio groups include methylthio,ethylthio, and the like. The term “alkylthio” also encompassescycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.“Arylthio” refers to aryl or heteroaryl groups. Alkylthio groups can besubstituted as defined above for alkyl groups.

The terms “alkenyl” and “alkynyl”, refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy,” as used herein, refers to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl is an ether or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, and —O— alkynyl. The terms“aroxy” and “aryloxy”, as used interchangeably herein, can berepresented by —O-aryl or O-heteroaryl, wherein aryl and heteroaryl areas defined below. The alkoxy and aroxy groups can be substituted asdescribed above for alkyl.

The terms “amine” and “amino” (and its protonated form) areart-recognized and refer to both unsubstituted and substituted amines,e.g., a moiety that can be represented by the general formula:

wherein R, R′, and R″ each independently represent a hydrogen, an alkyl,an alkenyl, —(CH₂)_(m)—R_(C) or R and R′ taken together with the N atomto which they are attached complete a heterocycle having from 4 to 8atoms in the ring structure; R_(C) represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In some embodiments, only one of R or R′ can bea carbonyl, e.g., R, R′ and the nitrogen together do not form an imide.In other embodiments, the term “amine” does not encompass amides, e.g.,wherein one of R and R′ represents a carbonyl. In further embodiments, Rand R′ (and optionally R″) each independently represent a hydrogen, analkyl or cycloakyl, an alkenyl or cycloalkenyl, or alkynyl. Thus, theterm “alkylamine” as used herein means an amine group, as defined above,having a substituted (as described above for alkyl) or unsubstitutedalkyl attached thereto, i.e., at least one of R and R′ is an alkylgroup.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R and R′ are as defined above.

As used herein, “Aryl” refers to C₅-C₁₀-membered aromatic, heterocyclic,fused aromatic, fused heterocyclic, biaromatic, or bihetereocyclic ringsystems. Broadly defined, “aryl”, as used herein, includes 5-, 6-, 7-,8-, 9-, and 10-membered single-ring aromatic groups that may includefrom zero to four heteroatoms, for example, benzene, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,pyrazine, pyridazine, pyrimidine, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as “arylheterocycles” or “heteroaromatics.” The aromatic ring can be substitutedat one or more ring positions with one or more substituents including,but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, andcombinations thereof. The term “aryl” includes phenyl.

The term “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (i.e., “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic ring or rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl. One or moreof the rings can be substituted as defined above for “aryl.”

The term “aralkyl,” as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aralkyloxy” can be represented by —O-aralkyl, wherein aralkylis as defined above.

The term “carbocycle,” as used herein, refers to an aromatic ornon-aromatic ring(s) in which each atom of the ring(s) is carbon.

“Heterocycle” or “heterocyclic,” as used herein, refers to a monocyclicor bicyclic structure containing 3-10 ring atoms, and in someembodiments, containing from 5-6 ring atoms, wherein the ring atoms arecarbon and one to four heteroatoms each selected from the followinggroup of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or isH, O, (C₁-C₁₀) alkyl, phenyl or benzyl, and optionally containing 1-3double bonds and optionally substituted with one or more substituents.Examples of heterocyclic rings include, but are not limited to,benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl,benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl,carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl,phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.Heterocyclic groups can optionally be substituted with one or moresubstituents at one or more positions as defined above for alkyl andaryl, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like. The terms “heterocycle”or “heterocyclic” can be used to describe a compound that can include aheterocycle or heterocyclic ring.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R and R′are as defined above. Where X is an oxygen and R or R′ is not hydrogen,the formula represents an “ester”. Where X is an oxygen and R is asdefined above, the moiety is referred to herein as a carboxyl group, andparticularly when R is a hydrogen, the formula represents a “carboxylicacid.” Where X is an oxygen and R′ is hydrogen, the formula represents a“formate.” In general, where the oxygen atom of the above formula isreplaced by sulfur, the formula represents a “thiocarbonyl” group. WhereX is a sulfur and R or R′ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R′ ishydrogen, the formula represents a “thioformate.” On the other hand,where X is a bond, and R is not hydrogen, the above formula represents a“ketone” group. Where X is a bond, and R is hydrogen, the above formularepresents an “aldehyde” group.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Exemplary heteroatoms include, but are notlimited to, boron, nitrogen, oxygen, phosphorus, sulfur, silicon,arsenic, and selenium. Heteroatoms, such as nitrogen, can have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms. It isunderstood that “substitution” or “substituted” includes the implicitproviso that such substitution is in accordance with permitted valenceof the substituted atom and the substituent, and that the substitutionresults in a stable compound, i.e., a compound that does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “nitro” refers to —NO₂; the term “halogen”designates —F, —Cl, —Br, or —I; the term “sulfhydryl” refers to —SH; theterm “hydroxyl” refers to —OH; and the term “sulfonyl” refers to —SO₂—.

As used herein, “carbamate” can be used to refer to a compound derivedfrom carbamic acid (NH₂COOH) and can include carbamate esters.“Carbamates” can have the general structure of:

Where R¹, R², and R³ can be any permissible substituent.

As used herein, “effective amount” can refer to the amount of acomposition described herein or pharmaceutical formulation describedherein that will elicit a desired biological or medical response of atissue, system, animal, plant, protozoan, bacteria, yeast or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician. The desired biological response can be modulation of boneformation and/or remodeling, including but not limited to modulation ofbone resorption and/or uptake of the BP conjugates, such as the BPquinolone conjugates, described herein. The effective amount will varydepending on the exact chemical structure of the composition orpharmaceutical formulation, the causative agent and/or severity of theinfection, disease, disorder, syndrome, or symptom thereof being treatedor prevented, the route of administration, the time of administration,the rate of excretion, the drug combination, the judgment of thetreating physician, the dosage form, and the age, weight, generalhealth, sex and/or diet of the subject to be treated. “Effective amount”can refer to the amount of a compositions described herein that iseffective to inhibit the growth of or reproduction of a microorganism,including but not limited to a bacterium or population thereof.“Effective amount” can refer to the amount of a compositions describedherein that is kill a microorganism, including but not limited to abacterium or population thereof. “Effective amount” can refer to theamount of a compositions described herein that is effective to treatand/or prevent osteomyelitis in a subject in need thereof.

As used herein, “therapeutic” generally can refer to treating, healing,and/or ameliorating a disease, disorder, condition, or side effect, orto decreasing in the rate of advancement of a disease, disorder,condition, or side effect. The term also includes within its scopeenhancing normal physiological function, palliative treatment, andpartial remediation of a disease, disorder, condition, side effect, orsymptom thereof.

As used herein, the terms “treating” and “treatment” can refer generallyto obtaining a desired pharmacological and/or physiological effect. Theeffect may be prophylactic in terms of preventing or partiallypreventing a disease, symptom or condition thereof.

As used herein, “synergistic effect,” “synergism,” or “synergy” refersto an effect arising between two or more molecules, compounds,substances, factors, or compositions that is greater than or differentfrom the sum of their individual effects.

As used herein, “additive effect” refers to an effect arising betweentwo or more molecules, compounds, substances, factors, or compositionsthat is equal to or the same as the sum of their individual effects.

The term “biocompatible”, as used herein, refers to a material thatalong with any metabolites or degradation products thereof that aregenerally non-toxic to the recipient and do not cause any significantadverse effects to the recipient. Generally speaking, biocompatiblematerials are materials which do not elicit a significant inflammatoryor immune response when administered to a patient.

As used herein, the term osteomyelitis can refer to acute or chronicosteomyelitis, and/or diabetic foot osteomyelitis, diabetic chronicosteomyelitis, prosthetic joint infections, periodontitis,peri-implantitis, osteonecrosis, and/or hematogenous osteomyelitisand/or other bone infections.

Discussion

Infectious bone disease, or osteomyelitis, is a major problem worldwidein human and veterinary medicine and can be devastating due to thepotential for limb-threatening sequelae and mortality. The treatmentapproach to osteomyelitis is mainly antimicrobial, and often long-term,with surgical intervention in many cases to control infection. Thecausative pathogens in most cases of long bone osteomyelitis arebiofilms of Staphylococcus aureus, which are bound to bone in contrastto their planktonic (free-floating) counterparts. Other bone infectionsare known to arise from a broad spectrum of both gram positive and gramnegative bacteria.

The biofilm-mediated nature of osteomyelitis is important in clinicaland experimental settings because many biofilm pathogens areuncultivable and exhibit an altered phenotype with respect to growthrate and antimicrobial resistance (as compared to their planktoniccounterparts). The difficulty in eradicating biofilms with conventionalantibiotics partly explains why the higher success rates ofantimicrobial therapy in general have not yet been realized fororthopedic infections, along with the development of resistant biofilmpathogens, the poor penetration of antimicrobial agents in bone, andadverse events related to systemic toxicity.

To overcome the many challenges associated with osteomyelitis treatment,there is increasing interest in drug delivery approaches usingbone-targeting conjugates to achieve higher or more sustained localtherapeutic concentrations of antibiotic in bone while minimizingsystemic exposure. Fluoroquinolone and non-fluoroquinolone antibioticsconjugated to bisphosphonates (BPs), for example osteoadsorptive BPs,represents a promising approach because of the long clinicaltrack-record of safety of each constituent, and their advantageousbiochemical properties. In early investigations of the fluoroquinolonefamily in this context, ciprofloxacin demonstrated the best binding andmicrobiological properties when bound to a BP. Ciprofloxacin has severaladvantages for repurposing in this context: it can be administeredorally or intravenously with relative bioequivalence, it has broadspectrum antimicrobial activity that includes the most commonlyencountered osteomyelitis pathogens, it demonstrates bactericidalactivity in clinically achievable doses, and it is the least expensivedrug in the fluoroquinolone family.

The specific bone-targeting properties of the BP family makes them idealcarriers for introducing antibiotics to bone in osteomyelitispharmacotherapy. BPs form strong bidentate and tridentate bonds withcalcium and as a result concentrate in hydroxyapatite (HA), particularlyat sites of active metabolism or infection and inflammation. BPs alsoexhibit exceptional stability against both chemical and biologicaldegradation. The concept of targeting ciprofloxacin to bone viaconjugation with a BP has been discussed in a number of reports over theyears.

Despite these positive attributes of BPs and fluoroquinolones, such asciprofloxacin, current attempts at generating prodrugs containing BPsand fluoroquinolones, such as ciprofloxacin, have been unsuccessful.Most attempts resulted in either systemically unstable prodrugs ornon-cleavable conjugates that were found to mostly inactivate eithercomponent of the conjugate by interfering with the pharmacophoricrequirements.

With the deficiencies of current BP fluoroquinolone conjugates in mind,described herein are BP quinolone conjugates that can contain a BP thatcan be releasably conjugated to a quinolone, such as ciprofloxacin. Inembodiments, the BP quinolone conjugate can selectively deliver aquinolone to bone, bone grafts, and or bone graft substitutes (i.e. cantarget bone, bone grafts, or bone graft substitutes) in a subject. Insome embodiments, the BP quinolone conjugate can release the quinolone.Also provided herein are methods of synthesizing BP quinolone conjugatesand methods of treating or preventing osteomyelitis or other boneinfections with one or more of the BP quinolone conjugates providedherein.

Other compositions, compounds, methods, features, and advantages of thepresent disclosure will be or become apparent to one having ordinaryskill in the art upon examination of the following drawings, detaileddescription, and examples. It is intended that all such additionalcompositions, compounds, methods, features, and advantages be includedwithin this description, and be within the scope of the presentdisclosure.

Bisphosphonate (BP) Quinolone Conjugates and Formulations Thereof

BP Quinolone Conjugates

Provided herein are BP quinolone conjugates and formulations thereof. ABP can be conjugated to a quinolone via a linker. In embodiments, thelinker is a releasable linker. The quinolone can be releasably attachedvia a linker to the BP. Thus, in some embodiments, the BP quinoloneconjugate can selectively deliver and release the quinolone at or nearbone, bone grafts, or bone graft substitutes (FIG. 13). In other words,the BP fluoroquinolone conjugate can provide targeted delivery offluoroquinolones to bone and/or the areas proximate to bone The BP ofthe BP quinolone conjugates provided herein can be any BP including butnot limited to, hydroxyl phenyl alkyl or aryl bisphosphonates, hydroxylphenyl (or aryl) alkyl hydroxyl bisphosphonates, amino phenyl(or aryl)alkyl bisphosphonates, amino phenyl(or aryl) alkyl hydroxylbisphosphonates, hydroxyl alkyl bisphosphonates, hydroxyl alkyl hydroxylbisphosphonates hydroxyl alkyl phenyl(or aryl) alkyl bisphosphonates,hydroxyl phenyl(or aryl) alkyl hydroxyl bisphosphonates, amino phenyl(oraryl) alkyl bisphosphonates, amino phenyl(or aryl) alkyl hydroxylbisphosphonates, hydroxyl alkyl bisphosphonates, hydroxyl alkyl hydroxylbisphosphonates (all of the former being further unsubstituted orsubstituted, etidronate, pamidronate, neridronate, olpadronate,alendronate, ibandronate, risedronate, zoledronate,hydroxymethylenebisphosphonate, and combinations thereof. Bisphosphonatemay also be substituted for phosphono phosphinic acid or phosphonocarboxylic acid. In embodiments, the BP can be pamidronate, alendronate,risedronate, zoledronate, minodronate, neridronate, etidronate, whichcan be unmodified or modified as described herein.

The BP can be modified to contain an alpha-hydroxy group (e.g.alpha-hydroxy modified risedronate and zoledronate, FIG. 29) Other BPscan be modified in the same way. In some embodiments, the BP can bemodified by substituting or removing the alpha-hydroxy group. (FIG. 30,e.g. p-PyrEBP). Removal or substitution of the alpha-hydroxyl group canreduce or eliminate the anti-resorptive effect of the BP as compared toan unmodified equivalent BP. As such, in some embodiments, the BPconjugates provided herein can contain a BP that lacks the alpha-hydroxygroup or has a substituted alpha-hydroxy group. Suitable substitutionsfor the alpha-hydroxy group can include, but are not limited to, H,alkyl, aryl, alkyl aryl. Further additional molecules conjugated to theBP can also affect the anti-resorptive effect. For example, when thequinolone and/or linker is coupled to the BP having a para-substitutedside change, the anti-resorptive effect can be significantly reduced oreliminated. In some embodiments, the BP can be modified to include bothan alpha hydroxyl deletion or substitution and a para-substituted sidechain.

In BPs containing an aryl or phenyl, the aryl or phenyl can besubstituted with a suitable substitutent at any position on the ring. Insome embodiments, the aryl or phenyl ring of the BP is substituted withone or more electron donating species (e.g. F, N, and Cl).

Non-pharmacologically active BP variants may also be used for thepurpose of fluoroquinolone delivery absent BP action.

The quinolone can be any quinolone, including but not limited toalatrofloxacin, amifloxacin, balofloxacin, besifloxacin, cadazolid,ciprofloxacin, clinafloxacin, danofloxacin, delafloxacin, difloxacin,enoxacin, enrofloxacin, finafloxacin, flerofloxacin, flumequine,gatifloxacin, gemifloxacin, grepafloxacin, ibafloxacin, JNJ-Q2,levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin,norfloxacin, ofloxacin, orbifloxacin, pazufloxacin, pefloxacin,pradofloxacin, prulifloxacin, rufloxacin, sarafloxacin, sitafloxacin,sparfloxacin, temafloxacin, tosufloxacin, trvafloxacin, zabofloxacin,nemonoxacin and any combination thereof. The quinolone can be afluoroquinolone.

The quinolone can have a generic structure according to Formula 1, whereR¹ can be substituents including alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl,carboxyl, substituted carboxyl, amino, substituted amino, amido,substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups, where R² can be substituents including alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy,phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio,substituted alkylthio, phenylthio, substituted phenylthio, arylthio,substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, sulfonyl, substituted sulfonyl,sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups, where R³ can be substituentsincluding alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl,alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy,substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano,substituted isocyano, carbonyl, substituted carbonyl, carboxyl,substituted carboxyl, amino, substituted amino, amido, substitutedamido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl,substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups, and where R⁴ can be substituents including alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy,phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio,substituted alkylthio, phenylthio, substituted phenylthio, arylthio,substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, amino, substitutedamino, amido, substituted amido, sulfonyl, substituted sulfonyl,sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups.

The BP can be conjugated to the fluoroquinolone via a releasable linker.In some embodiments the releasable linker can be a phenyl carbamatelinker. The releasable linker can be an aryl carbamate linker. In someembodiments the linker can be an aryl thiocarbamate linker. In someembodiments the linker can be a phenyl thiocarbamate linker. In someembodiments the thiocarbamate linker can be an O-thiocarbamate linker.In some embodiments, the thiocarbamate linker can be an S-thiocarbamatelinker. In some embodiments, the linker can be a carbonate linker. Insome embodiments the linker can be a urea linker. In some embodiments,the linker can be an aryl dithiocarbamate linker.

In various aspects, an alpha-OH containing BP can be conjugated to thequinolone, such as a quinolone (including any fluoroquinolone), at ageminal OH group on the BP as shown below. In some aspects thequinolone, such as a fluoroquinolone or other quinolone as describedherein, can be indirectly conjugated via a linker, such as describedherein, at the geminal OH group of the BP.

conjugates between alpha-OH containing BP and fluoroquinolone In someaspects, the compound can have a formula according to Formula (41) orFormula (43)

BP Quinolone Conjugate Pharmaceutical Formulations

Also described herein are formulations, including pharmaceuticalformulations, which can contain an amount of a BP quinolone conjugatedescribed elsewhere herein. The amount can be an effective amount. Theamount can be effective to inhibit the growth and/or reproduction of abacterium. The amount can be effective to kill a bacterium.Formulations, including pharmaceutical formulations can be formulatedfor delivery via a variety of routes and can contain a pharmaceuticallyacceptable carrier. Techniques and formulations generally can be foundin Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton,Pa. (20^(th) Ed., 2000), the entire disclosure of which is hereinincorporated by reference. For systemic administration, an injection isuseful, including intramuscular, intravenous, intraperitoneal, andsubcutaneous. For injection, the therapeutic compositions of theinvention can be formulated in liquid solutions, for example inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the BP quinolone conjugates and/or componentsthereof can be formulated in solid form and redissolved or suspendedimmediately prior to use. Lyophilized forms are also included.Formulations, including pharmaceutical formulations, of the BP quinoloneconjugates can be characterized as being at least sterile andpyrogen-free. These formulations include formulations for human andveterinary use.

Suitable pharmaceutically acceptable carriers include, but are notlimited to water, salt solutions, alcohols, gum arabic, vegetable oils,benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such aslactose, amylose or starch, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid esters, hydroxylmethylcellulose, and polyvinyl pyrrolidone, which do not deleteriouslyreact with BP quinolone conjugate.

The pharmaceutical formulations can be sterilized, and if desired, mixedwith auxiliary agents, such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, flavoring and/or aromatic substances, and the likewhich do not deleteriously react with the BP quinolone conjugate.

Another formulation includes the addition of BP quinolone conjugates tobone graft material or bone void fillers for the prevention or treatmentof osteomyelitis, peri-implantitis or peri-prosthetic infections, andfor socket preservation after dental extractions.

The pharmaceutical formulations can be formulated to be compatible withtheir intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerin, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Formulations, including pharmaceutical formulations, suitable forinjectable use can include sterile aqueous solutions (where watersoluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. Forintravenous administration, suitable carriers can include physiologicalsaline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). Injectable pharmaceutical formulationscan be sterile and can be fluid to the extent that easy syringabilityexists. Injectable pharmaceutical formulations can be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, a pharmaceutically acceptable polyol like glycerol,propylene glycol, liquid polyethylene glycol, and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In someembodiments, it can be useful to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride inthe composition.

Sterile injectable solutions can be prepared by incorporating any of BPquinolone conjugates described herein in an amount in an appropriatesolvent with one or a combination of ingredients enumerated herein, asrequired, followed by filtered sterilization. Generally, dispersions canbe prepared by incorporating BP quinolone conjugate into a sterilevehicle which contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterile powdersfor the preparation of sterile injectable solutions, examples of usefulpreparation methods are vacuum drying and freeze-drying which yields apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated can be used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fluidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the BP quinolone conjugates can beformulated into ointments, salves, gels, or creams as generally known inthe art. In some embodiments, the BP quinolone conjugates can be appliedvia transdermal delivery systems, which can slowly release the BPquinolone conjugates for percutaneous absorption. Permeation enhancerscan be used to facilitate transdermal penetration of the active factorsin the conditioned media. Transdermal patches are described in forexample, U.S. Pat. Nos. 5,407,713; 5,352,456; 5,332,213; 5,336,168;5,290,561; 5,254,346; 5,164,189; 5,163,899; 5,088,977; 5,087,240;5,008,110; and 4,921,475.

For oral administration, a formulation as described herein can bepresented as capsules, tablets, powders, granules, or as a suspension orsolution. The formulation can contain conventional additives, such aslactose, mannitol, cornstarch or potato starch, binders, crystallinecellulose, cellulose derivatives, acacia, cornstarch, gelatins,disintegrators, potato starch, sodium carboxymethylcellulose, dibasiccalcium phosphate, anhydrous or sodium starch glycolate, lubricants,and/or or magnesium stearate.

For parenteral administration (i.e., administration through a routeother than the alimentary canal), the formulations described herein canbe combined with a sterile aqueous solution that is isotonic with theblood of the subject. Such a formulation can be prepared by dissolvingthe active ingredient (e.g. the BP quinolone conjugate) in watercontaining physiologically-compatible substances, such as sodiumchloride, glycine and the like, and having a buffered pH compatible withphysiological conditions, so as to produce an aqueous solution, thenrendering the solution sterile. The formulation can be presented in unitor multi-dose containers, such as sealed ampoules or vials. Theformulation can be delivered by injection, infusion, or other meansknown in the art.

For transdermal administration, the formulations described herein can becombined with skin penetration enhancers, such as propylene glycol,polyethylene glycol, isopropanol, ethanol, oleic acid,N-methylpyrrolidone and the like, which increase the permeability of theskin to the nucleic acid vectors of the invention and permit the nucleicacid vectors to penetrate through the skin and into the bloodstream. Theformulations and/or compositions described herein can be furthercombined with a polymeric substance, such as ethylcellulose,hydroxypropyl cellulose, ethylene/vinyl acetate, polyvinyl pyrrolidone,and the like, to provide the composition in gel form, which can bedissolved in a solvent, such as methylene chloride, evaporated to thedesired viscosity and then applied to backing material to provide apatch.

For inclusion in bone graft substitutes or bone void fillers to preventlocal post-operative infection or graft failure after surgery, and toprovide sustained local release of antibiotic at the graft site, theformulations described herein can be combined with any xenograft(bovine), autograft (self) or allograft (cadaver) material or syntheticbone substitute. For example, a powder formulation can be premixed bythe treating surgeon or clinician bedside/chairside with any existingbone graft substitute on the market or with an autologous graft. Thisformulation can be further combined with any previously describedformulation, and can be combined with products containinghydroxyapatites, tricalcium phosphates, collagen, aliphatic polyesters(poly(lactic) acids (PLA), poly(glycolic) acids (PGA), andpolycaprolactone (PCL), polyhydroxybutyrate (PHB), methacrylates,polymethylmethacrylates, resins, monomers, polymers, cancellous boneallografts, human fibrin, platelet rich plasma, platelet rich fibrin,plaster of Paris, apatite, synthetic hydroxyapatite, corallinehydroxyapatite, wollastonite (calcium silicate), calcium sulfate,bioactive glasses, ceramics, titanium, devitalized bone matrix,non-collagenous proteins, collagen, and autolyzed antigen extractedallogenic bone. In this embodiment the bone graft material combined withBP quinolone conjugate can be in the formulation of a paste, powder,putty, gel, hydrogel, matrix, granules, particles, freeze-dried powder,freeze-dried bone, demineralized freeze-dried bone, fresh orfresh-frozen bone, corticocancellous mix, pellets, strips, plugs,membranes, lyophilized powder reconstituted to form wet paste,spherules, sponges, blocks, morsels, sticks, wedges, cements, oramorphous particles; many of these may also be in injectableformulations or as a combination of two or more aforementionedformulations (e.g. injectable paste with sponge).

In another embodiment, BP-quinolone conjugate can be combined withfactor-based bone grafts containing natural or recombinant growthfactors, such as transforming growth factor-beta (TGF-beta),platelet-derived growth factor (PDGF), fibroblast growth factors (FGF),and/or bone morphogenic protein (BMP). In another embodiment, BPquinolone conjugate can be combined with cell-based bone grafts used inregenerative medicine and dentistry including embryonic stem cellsand/or adults stem cells, tissue-specific stem cells, hematopoietic stemcells, epidermal stem cells, epithelial stem cells, gingival stem cells,periodontal ligament stem cells, adipose stem cells, bone marrow stemcells, and blood stem cells. Therefore, a bone graft with the propertyof osteoconduction, osteoinduction, osteopromotion, osteogenesis, or anycombination thereof can be combined with BP quinolone conjugate forclinical or therapeutic use.

Dosage Forms

The BP quinolone conjugates, compounds, and formulations thereof,described herein can be provided in unit dose form such as a tablet,capsule, single-dose injection or infusion vial, or as a predetermineddose for mixing with bone graft material as in formulations describedabove. Where appropriate, the dosage forms described herein can bemicroencapsulated. The dosage form can also be prepared to prolong orsustain the release of any ingredient. In some embodiments, thecomplexed active agent can be the ingredient whose release is delayed.In other embodiments, the release of an auxiliary ingredient is delayed.Suitable methods for delaying the release of an ingredient include, butare not limited to, coating or embedding the ingredients in material inpolymers, wax, gels, and the like. Delayed release dosage formulationscan be prepared as described in standard references such as“Pharmaceutical dosage form tablets,” eds. Liberman et. al. (New York,Marcel Dekker, Inc., 1989), “Remington—The science and practice ofpharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, Md.,2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6thEdition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). Thesereferences provide information on excipients, materials, equipment, andprocesses for preparing tablets and capsules and delayed release dosageforms of tablets and pellets, capsules, and granules. The delayedrelease can be anywhere from about an hour to about 3 months or more.

Coatings may be formed with a different ratio of water soluble polymer,water insoluble polymers, and/or pH dependent polymers, with or withoutwater insoluble/water soluble non polymeric excipient, to produce thedesired release profile. The coatings can be either performed on thedosage form (matrix or simple) which includes, but is not limited to,tablets (compressed with or without coated beads), capsules (with orwithout coated beads), beads, particle compositions, “ingredient as is”formulated as, but not limited to, suspension form or as a sprinkledosage form.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate, and hydroxypropyl methylcellulose acetate succinate;polyvinyl acetate phthalate, acrylic acid polymers and copolymers, andmethacrylic resins that are commercially available under the trade nameEUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, andpolysaccharides.

Effective Amounts

The formulations can contain an effective amount of a BP quinoloneconjugate or compound (effective for inhibiting and/or killing abacterium) described herein. In some embodiments, the effective amountranges from about 0.001 pg to about 1,000 g or more of the BP quinoloneconjugate described herein. In some embodiments, the effective amount ofthe BP quinolone conjugate described herein can range from about 0.001mg/kg body weight to about 1,000 mg/kg body weight. In yet otherembodiments, the effective amount of the BP quinolone conjugate canrange from about 1% w/w to about 99% or more w/w, w/v, or v/v of thetotal formulation. In some embodiments, the effective amount of the BPquinolone conjugate is effective at killing a bacterium that is thecausative agent of osteomyelitis and all its subtypes (e.g. diabeticfoot osteomyelitis), jaw osteonecrosis, and periodontitis including, butnot limited to any strain or species of Staphylococcus, Pseudomonas,Aggregatibacter, Actinomyces, Streptococcus, Haemophilus, Salmonella,Serratia, Enterobacter, Fusobacterium, Bacteroides, Porphyromonas,Prevotella, Veillonella, Campylobacter, Peptostreptococcus, Eikenella,Treponema, Dialister, Micromonas, Yersinia, Tannerella, and Escherichia.

Methods of Using the BP Quinolone Conjugates

An amount, including an effective amount, of the BP quinoloneconjugates, compounds, and formulations thereof, described herein can beadministered to a subject in need thereof. In some embodiments thesubject in need thereof can have a bone infection, disease, disorder, ora symptom thereof. In some embodiments, the subject in need thereof canbe suspected of having or is otherwise predisposed to having a boneinfection, disease, disorder, or a symptom thereof. In some embodiments,the subject in need thereof may be at risk for developing anosteomyelitis, osteonecrosis, peri-prosthetic infection, and/orperi-implantitis. In embodiments, the disease or disorder can beosteomyelitis and all its subtypes, osteonecrosis, peri-implantitis orperiodontitis. In some embodiments the subject in need thereof has abone that is infected with a microorganism, such as a bacteria. In someembodiments, the bacteria can be any strain or species ofStaphylococcus, Pseudomonas, Aggregatibacter, Actinomyces,Streptococcus, Haemophilus, Salmonella, Serratia, Enterobacter,Fusobacterium, Bacteroides, Porphyromonas, Prevotella, Veillonella,Campylobacter, Peptostreptococcus, Eikenella, Treponema, Dialister,Micromonas, Yersinia, Tannerella, and Escherichia. In some embodiments,the bacteria can form biofilms. In some embodiments, osteomyelitis canbe treated in a subject in need thereof by administering an amount, suchas an effective amount, of BP quinolone conjugate or formulation thereofdescribed herein to the subject in need thereof. In some embodiments,the compositions and compounds provided herein can be used inosteonecrosis treatment and/or prevention, distraction osteogenesis,cleft repair, repair of critical supra-alveolar defects, jawbonereconstruction, and any other reconstructions or repair of a bone and/orjoint.

Administration of the BP quinolone conjugates is not restricted to asingle route, but can encompass administration by multiple routes. Forinstance, exemplary administrations by multiple routes include, amongothers, a combination of intradermal and intramuscular administration,or intradermal and subcutaneous administration. Multiple administrationscan be sequential or concurrent. Other modes of application by multipleroutes will be apparent to the skilled artisan.

The pharmaceutical formulations can be administered to a subject by anysuitable method that allows the agent to exert its effect on the subjectin vivo. For example, the formulations and other compositions describedherein can be administered to the subject by known procedures including,but not limited to, by oral administration, sublingual or buccaladministration, parenteral administration, transdermal administration,via inhalation, via nasal delivery, vaginally, rectally, andintramuscularly. The formulations or other compositions described hereincan be administered parenterally, by epifascial, intracapsular,intracutaneous, subcutaneous, intradermal, intrathecal, intramuscular,intraperitoneal, intrasternal, intravascular, intravenous,parenchymatous, and/or sublingual delivery. Delivery can be byinjection, infusion, catheter delivery, or some other means, such as bytablet or spray. Delivery can also be by a carrier such ashydroxyapatite or bone in the case of anti-infective bone graft materialat a surgical site. Delivery can be via attachment or other associationwith a bone graft material.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of the present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure.

Example 1

Introduction

Infectious bone disease, or osteomyelitis, is a major problem worldwidein human and veterinary medicine and can be devastating due to thepotential for limb-threatening sequelae and mortality (Lew, et al.,Osteomyelitis. Lancet 2004; 364:369-79; Desrochers, et al, Limbamputation and prosthesis. Vet Clin North Am Food Anim Pract 2014;30:143-55; Stoodley, et al., Orthopaedic biofilm infections. Curr OrthopPract 2011; 22:558-63; Huang, et al., Chronic osteomyelitis increaseslong-term mortality risk in the elderly: a nationwide population-basedcohort study. BMC Geriatr 2016; 16:72). The treatment approach toosteomyelitis is mainly antimicrobial, and often long-term, withsurgical intervention in many cases to control infection. The causativepathogens in most cases of long bone osteomyelitis are biofilms ofStaphylococcus aureus; by definition these microbes are bound to bone(FIG. 1) in contrast to their planktonic (free-floating) counterparts(Wolcott, et al., Biofilms and chronic infections. J Am Med Assoc 2008;299:2682-2684).

The biofilm-mediated nature of osteomyelitis is important in clinicaland experimental settings because many biofilm pathogens areuncultivable and exhibit an altered phenotype with respect to growthrate and antimicrobial resistance (as compared to their planktoniccounterparts) (Junka, et al., Microbial biofilms are able to destroyhydroxyapatite in the absence of host immunity in vitro. J OralMaxillofac Surg 2015; 73:451-64; Herczegh, et al., Osteoadsorptivebisphosphonate derivatives of fluoroquinolone antibacterials. J Med Chem2002; 45:2338-41). The difficulty in eradicating biofilms withconventional antibiotics partly explains why the higher success rates ofantimicrobial therapy in general have not yet been realized fororthopedic infections, along with the development of resistant biofilmpathogens, the poor penetration of antimicrobial agents in bone, andadverse events related to systemic toxicity (Buxton, et al.,Bisphosphonate-ciprofloxacin bound to Skelite is a prototype forenhancing experimental local antibiotic delivery to injured bone. Br JSurg 2004; 91:1192-6).

To overcome the many challenges associated with osteomyelitis treatment,there is increasing interest in drug delivery approaches usingbone-targeting conjugates to achieve higher or more sustained localtherapeutic concentrations of antibiotic in bone while minimizingsystemic exposure (Panagopoulos, et al., Local Antibiotic DeliverySystems in Diabetic Foot Osteomyelitis: Time for One Step Beyond? Int JLow Extrem Wounds 2015; 14:87-91; Puga, et al., Hot meltpoly-epsilon-caprolactone/poloxamine implantable matrices for sustaineddelivery of ciprofloxacin. Acta biomaterialia 2012; 8:1507-18).Fluoroquinolone antibiotics conjugated to osteoadsorptivebisphosphonates (BPs) represents a promising approach because of thelong clinical track-record of safety of each constituent, and theiradvantageous biochemical properties (Buxton, et al.,Bisphosphonate-ciprofloxacin bound to Skelite is a prototype forenhancing experimental local antibiotic delivery to injured bone. Br JSurg 2004; 91:1192-6). In early investigations of the fluoroquinolonefamily in this context, ciprofloxacin demonstrated the best binding andmicrobiological properties when bound to BP (Herczegh, et al.,Osteoadsorptive bisphosphonate derivatives of fluoroquinoloneantibacterials. J Med Chem 2002; 45:2338-41). Ciprofloxacin has severaladvantages for repurposing in this context: it can be administeredorally or intravenously with relative bioequivalence, it has broadspectrum antimicrobial activity that includes the most commonlyencountered osteomyelitis pathogens, it demonstrates bactericidalactivity in clinically achievable doses, and it is the least expensivedrug in the fluoroquinolone family (Houghton, et al., Linkingbisphosphonates to the free amino groups in fluoroquinolones:preparation of osteotropic prodrugs for the prevention of osteomyelitis.J Med Chem 2008; 51:6955-69).

The specific bone-targeting properties of the BP family makes them idealcarriers for introducing antibiotics to bone in osteomyelitispharmacotherapy (Zhang S, et al., ‘Magic bullets’ for bone diseases:progress in rational design of bone-seeking medicinal agents. Chem SocRev 2007; 36:507-31). BPs form strong bidentate and tridentate bondswith calcium and as a result concentrate in hydroxyapatite (HA),particularly at sites of active metabolism or infection and inflammation(Cheong, et al., Bisphosphonate uptake in areas of tooth extraction orperiapical disease. J Oral Maxillofac Surg 2014; 72:2461-8). BPs alsoexhibit exceptional stability against both chemical and biologicaldegradation (Russell, et al., Mechanisms of action of bisphosphonates:similarities and differences and their potential influence on clinicalefficacy. Osteoporos Int 2008; 19:733-59). The concept of targetingciprofloxacin to bone via conjugation with BP has been discussed in anumber of reports over the years (David, et al.,Methylene-bis[(aminomethyl)phosphinic acids]: synthesis, acid-base andcoordination properties. Dalton Trans 2013; 42:2414-22; Fardeau, et al.,Synthesis and antibacterial activity of catecholate-ciprofloxacinconjugates. Bioorg Med Chem 2014; 22:4049-60; EUCAST: European Committeeon Antimicrobial Susceptibility Testing breakpoint tables forinterpretation of MICs and zone diameters. 2015.http://www.eucast.org/fileadmin/src/media/PDFs/EUC; CLSI. M100-S25performance standards for antimicrobial susceptibility testing,Twenty-fifth informational supplement, 2015; Tanaka, et al.,Bisphosphonated fluoroquinolone esters as osteotropic prodrugs for theprevention of osteomyelitis. Bioorg Med Chem 2008; 16:9217-29;McPherson, et al., Synthesis of osteotropic hydroxybisphosphonatederivatives of fluoroquinolone antibacterials. Eur J Med Chem 2012;47:615-8). However, early attempts resulted in either systemicallyunstable prodrugs or non-cleavable conjugates that were found to mostlyinactivate either component of the conjugate by interfering with thepharmacophoric requirements. In the fluoroquinolone field a prominentexample was described by Herczegh et al where significant gram-positiveantibacterial properties of the ciprofloxacin constituent were lost onconjugation with a stable BP-linked congener (Herczegh, et al.,Osteoadsorptive bisphosphonate derivatives of fluoroquinoloneantibacterials. J. Med. Chem 2002; 45:2338-41). Subsequentinvestigations in this field have elucidated that these conjugates alonecannot exert significant antimicrobial effects without cleavage of theparent antibiotic (Herczegh, et al., Osteoadsorptive bisphosphonatederivatives of fluoroquinolone antibacterials. J. Med. Chem 2002;45:2338-41; Houghton, et al., Linking bisphosphonates to the free aminogroups in fluoroquinolones: preparation of osteotropic prodrugs for theprevention of osteomyelitis. J. Med. Chem 2008; 51:6955-69). Houghton etal, for example, synthesized and tested various BP-fluoroquinoloneconjugates and found that phenylpropanone and acyloxyalkyl carbamategatifloxacin prodrugs were possibly able to regenerate the parent drugonce bound to bone, and thus demonstrated greater antimicrobial activitythan simple conjugates such as bisphosphonoethyl, bisphosphonopropionyland amide derivatives which were unable to release the antibiotic(Houghton, et al., Linking bisphosphonates to the free amino groups influoroquinolones: preparation of osteotropic prodrugs for the preventionof osteomyelitis. J. Med. Chem 2008; 51:6955-69).

Taken together, the research findings in this field to date indicatethat BP-fluoroquinolone antimicrobial activity is complex and is relatedto the specific strain of pathogen tested, the choice of antibiotic andcovalently bound BP moiety, the tether length between the twoconstituents, the bone binding affinity of the BP, theadsorption-desorption equilibria of the BP, and the stability/labilityand kinetics of the linkage scheme used for conjugation (Herczegh, etal., Osteoadsorptive bisphosphonate derivatives of fluoroquinoloneantibacterials. J. Med. Chem 2002; 45:2338-41; Houghton, et al., Linkingbisphosphonates to the free amino groups in fluoroquinolones:preparation of osteotropic prodrugs for the prevention of osteomyelitis.J. Med. Chem 2008; 51:6955-69; Tanaka, et al., Bisphosphonatedfluoroquinolone esters as osteotropic prodrugs for the prevention ofosteomyelitis. Bioorg Med Chem 2008; 16:9217-29; McPherson, et al.,Synthesis of osteotropic hydroxybiphosphonate derivatives offluoroquinolone antibacterials. Eur J. Med Chem 2012:47:615-8).Therefore, accumulating evidence suggests that a ‘target and release’linker strategy may offer more opportunities for optimization andsuccess in this context. We thus hypothesized that conjugation ofciprofloxacin to a phenyl BP moiety, through metabolically hydrolyzablecarbamate linkers, should mitigate the problems seen with antibioticbone dosing in osteomyelitis pharmacotherapy. The cleavable carbamatelinkage and structural motif is a key functionality in many drugsdesigned for target and release in specific tissues, and conferspharmacokinetic advantages such as stability in serum and lability atinfected bone surfaces in the presence of an acidic and enzymaticenvironment (Ossipov, et al., Bisphosphonate-modified biomaterials fordrug delivery and bone tissue engineering. Expert Opin Drug Deliv 2015;12:1443-58; Guo, et al., pH-triggered intracellular release fromactively targeting polymer micelles. Biomaterials 2013; 34:4544-54;Ghosh, et al., Organic carbamates in drug design and medicinalchemistry. J Med Chem 2015; 58:2895-940).

One recent apparent success utilizing a bone-targeting and releasestrategy has been observed where Morioka et al designed an estradiolanalog to target and release at bone, using a cleavable variant(carbamate) of the more stable amide peptide bond (Morioka, et al.,Design, synthesis, and biological evaluation of novelestradiol-bisphosphonate conjugates as bone-specific estrogens. BioorgMed Chem 2010; 18:1143-8). Several versions of this linkage wereattempted before the identification of a pharmacologically activevariant (phenyl carbamate). Importantly, they demonstrated that a 1000×lower single dose of a similarly linked BP-estradiol conjugate produceda similar effect on bone to that of estradiol dosed alone (Morioka, etal., Design, synthesis, and biological evaluation of novelestradiol-bisphosphonate conjugates as bone-specific estrogens. BioorgMed Chem 2010; 18:1143-8). The conjugate also provided a largertherapeutic index or improved safety, as there were minimal effects inuterine tissue. Pharmacokinetic studies completed by Arns et al are inagreement with this dramatic enhancement of potency with a phenylcarbamate linked BP-prostaglandin (Arns, et al., Design and synthesis ofnovel bone-targeting dual-action pro-drugs for the treatment andreversal of osteoporosis. Bioorg Med Chem 2012; 20:2131-40). A syntheticexample of this approach in the antimicrobial field is reported for themacrolide class; however, only alkyl carbamates were explored and lackof further success suggests that target and release strategies arelikely chemical class-dependent (taking into considerationcompatibilities of the functional groups of each component) as well asbiochemical target-dependent, and the design for any particular chemicalclass must be customized for its use (Tanaka, et al., Synthesis and invitro evaluation of bisphosphonated glycopeptide prodrugs for thetreatment of osteomyelitis. Bioorg Med Chem Lett 2010; 20:1355-9).

This Example demonstrates a phenyl carbamate BP-ciprofloxacin conjugateand systematical evaluation of its antimicrobial activity in vitroagainst common osteomyelitis pathogens, and assessed in vivo safety andefficacy in an animal model of peri-implant osteomyelitis. Importantly,the in vitro and in vivo studies presented herein are predicated onbiofilm models and methodology in addition to planktonic cultures, whichhas not been performed to date in this field and which should providefor greater clinical relevance. The present study specifically addressesan unmet medical need in the treatment of infectious bone disease, andthus has been designed for translational significance.

Results and Discussion

Chemistry:

The overall synthetic route for the one BP-ciprofloxacin conjugate (BCC,compound 6) is shown in Schemes in FIGS. 2A-2B. As a starting point ourproject team identified the inert 4-hydroxyphenylethylidene BP for thisconjugation. The rationale for this BP design was to retain thebone-seeking ability of the BP moiety while suppressing its unneededantiresorptive activity, enabling us to minimize confounders and focusuniquely on evaluating the antimicrobial effect due to the parentciprofloxacin compound. BP ligands can be designed to haveantiresorptive functionality (of varying potency) if needed to provide adual-action effect of bone tissue protection in addition toantimicrobial effects at the anatomic site of infection. We also chosethis phenyl BP with consideration to bone binding affinity and tetherlength, as previous studies have demonstrated that weak binding affinitydecreases targeting efficiency and that lengthening the distance betweenthe fluoroquinolone and the BP functionality can decrease the rate ofhydrolysis and regeneration of the parent compound (Houghton, et al.,Linking bisphosphonates to the free amino groups in fluoroquinolones:preparation of osteotropic prodrugs for the prevention of osteomyelitis.J. Med. Chem. 2008; 51:6955-69; Tanaka, et al., Bisphosphonatedfluoroquinolone esters as osteotropic prodrugs for the prevention ofosteomyelitis, Bioorg Med Chem 2008, 16:9217-29; McPherson, et al.,Synthesis of osteotropic hydroxybisphosphonate derivatives offluoroquinolone antibacterials, Eur J Med Chem 2012, 47:615-8). Mostimportantly, we believed the use of an aryl carbamate as a linker mightoffer optimized stability in plasma and adequate release on bone forthis biochemical target relative to previous BP-F quinolone conjugates.Accordingly, the tetraethyl ester of 4-hydroxyphenylethylidene BP (4)was prepared as described previously (David, et al.,Methylene-bis[(aminomethyl)phosphinic acids]: synthesis, acid-base andcoordination properties. Dalton Trans 2013; 42:2414-22). The phenolgroup of BP (4) was then activated with p-nitrophenyl chloroformate toform compound (5) for conjugation with protected ciprofloxacin (7)(Fardeau, et al., Synthesis and antibacterial activity ofcatecholate-ciprofloxacin conjugates. Bioorg Med Chem 2014; 22:4049-60).Ciprofloxacin (6) was protected with a benzyl (Bn) group via aDi-t-butyl dicarbonate (Boc₂O) reaction. Final deprotection of theconjugate (8) with hydrogenolysis and bromotrimethylsilane (TMSBr) leadto our first fluoroquinolone phenyl carbamate BP-ciprofloxacin prodrug(9) ready for biochemical and antimicrobial evaluations.

Microbiology:

The first set of investigations we undertook were aimed at evaluatingthe antimicrobial activity of the conjugate in standard laboratoryplanktonic culture systems against a panel of 14 S. aureus clinicalstrains associated with bone infections (methicillin-sensitive: MSSA andmethicillin-resistant: MRSA). Following EUCAST (European Committee onAntimicrobial Susceptibility Testing) guidelines, results from discdiffusion inhibition zone assays revealed diameters ranging from 25-40mm (mean 31.5, SD±5), and every strain demonstrated antimicrobialsensitivity according to EUCAST breakpoints (EUCAST: European Committeeon Antimicrobial Susceptibility Testing breakpoint tables forinterpretation of MICs and zone diameters. 2015.http://www.eucast.org/fileadmin/src/media/PDFs/EUC). MIC results forBP-ciprofloxacin tested against all 14 strains using microdilutionmethodology are shown in FIG. 3. MICs for the parent compoundciprofloxacin alone were determined concurrently for reference (whichshows Table 1) and were found to be consistent with established clinicalbreakpoints.²⁶ It has already been established that prodrugs in thisclass lack significant antibacterial activity of their own, and that anyBP-related antimicrobial effect is negligible, therefore release of theparent drug is a prerequisite for observing any appreciableantimicrobial activity such as that reported here (Houghton, et al.,Linking bisphosphonates to the free amino groups in fluoroquinolones:preparation of osteotropic prodrugs for the prevention of osteomyelitis.J. Med. Chem. 2008, 51:6955-69).

The AST and MIC data indicate that against planktonic S. aureuspathogens both the conjugate and ciprofloxacin have bactericidalactivity, and that conjugation impacts ciprofloxacin antimicrobialactivity in vitro with slightly greater concentrations of conjugaterequired to reach MIC than ciprofloxacin alone. This is anticipatedsince it is well-established that conjugation is based on chemicalmodification of both BP and the antibiotic that has to be delivered tobone; as a result, properties of the parent drug including itstherapeutic effect can be altered by such modification. Our results arealso consistent with previous literature in this field indicating thatsuccessful and functional conjugates retain the antibacterial activityof the parent compound, albeit at a slightly lower level (Herczegh, etal., Osteoadsorptive bisphosphonate derivatives of fluoroquinoloneantibacterials. J. Med. Chem 2002, 45:2338-41; Houghton, et al., Linkingbisphosphonates to the free amino groups in fluoroquinolones:preparation of osteotropic prodrugs for the prevention of osteomyelitis.J. Med. Chem 2008, 51: 6955-69; Zhang, et al., ‘Magic Bullets’ for bonediseases: progress in rational design of bone-seeking medicinal agents).Importantly, in the therapeutic context of osteomyelitis the pathogensare not planktonic (as in these standard assays) but rather biofilm, andbound to bone as a substrate, so the enhanced bone targeting property ofthe BP-ciprofloxacin conjugate should provide more than adequateconcentrations of antibiotic for antimicrobial effect at bone and thusgreater efficacy (as forthcoming biofilm-relevant in vitro and in vivodata support).

Because microbiological media used for in vitro antimicrobial testinghas proteins, carbohydrates, enzymes and salts/metals, the potentialexists for degradation, denaturation or chelation of BP-ciprofloxacinduring antimicrobial testing. This could adversely impact antibioticactivity and be unrelated to the chemical conjugation itself. Based onour AST and MIC results and demonstrable antimicrobial efficacy of theconjugate this is highly unlikely to any significant extent.Nonetheless, we sought to objectively assess BP-ciprofloxacin stabilityby introducing the conjugate to trypticase soy broth microbiologicalmedia and conducting quantitative spectroscopic analysis as shown inFIG. 4. Results indicated excellent stability of the antimicrobial withno evidence of degradation or denaturation in microbiological mediaafter 24 hrs. Therefore, microbiological media likely has little to noadverse effect on conjugate activity and efficacy.

Having established the antimicrobial efficacy and chemical stability ofthe conjugate, we next sought to evaluate HA binding ability. When weadded HA spherules to our microbiological media and then introducedBP-ciprofloxacin at various concentrations similar to those used in ourantimicrobial testing, quantitative spectroscopic analysis ofsupernatant (without HA spherules) confirmed significant adsorption andretention of the conjugate by HA (FIG. 5). These results are consistentwith previously reported analogs in this class containing BP moietieswith similar bone affinities (Tanaka, et al., Bisphosphonatedfluoroquinolone esters as osteotropic prodrugs for the prevention ofosteomyelitis. Bioorg Med Chem 2008; 16:9217-29; McPherson, et al.,Synthesis of osteotropic hydroxybisphosphonate derivatives offluoroquinolone antibacterials. Eur J Med Chem 2012; 47:615-8). Boneadsorption also appeared to be a concentration-dependent phenomenon.

We then selected the S. aureus strain ATCC-6538 for further testingbecause it demonstrated the least susceptibility and poorest MIC profileto both ciprofloxacin and the conjugate (FIG. 3) as compared to othertested strains. This strain is also a well-known and robust biofilmforming pathogen as compared to other tested strains. Consequently, wecould test and optimize our conjugate against the most virulent pathogento limit bias and overestimated results, while also facilitating thetesting of antimicrobial activity in biofilm-based and clinicallyrelevant models. So, we performed AST on planktonic S. aureus strainATCC-6538 with BP-ciprofloxacin under both acidic and basic conditionsto assess the effect of pH on conjugate activity. Quantitative resultsfrom standard microdilution methodology indicated that under acidicconditions antimicrobial activity was improved overall, and the MIC⁵⁰was reached at half the conjugate concentration required to reach MIC⁵⁰under basic conditions (FIG. 6). This could be useful for clinicalosteomyelitis applications where biofilm pathogens along with hostinflammation and osteoclastogenesis produce an acidic local milieu.Other investigators have suggested, however, that although the localacidity brought on by infecting organisms and inflammation might beassociated with some drug release in bone, the efficiency of such aprocess in providing a sufficient concentration of the antimicrobialagent is doubtful, and that prodrug design and conjugation scheme likelyplay a greater role (Houghton, et al., Linking bisphosphonates to thefree amino groups in fluoroquinolones: preparation of osteotropicprodrugs for the prevention of osteomyelitis. J. Med. Chem 2008;51:6955-69). Finally, AST data also indicated that MICs forciprofloxacin and the conjugate were equivalent to their meanbactericidal concentrations (MBCs), respectively.

Next, time-kill assays were performed with the conjugate according toCLSI (Clinical Laboratory Standards Institute) methods and resultsindicated that the conjugate was bactericidal at the previouslyestablished MIC for methicillin-susceptible (ATCC-6538) andmethicillin-resistant (MR4-CIPS) isolates of planktonic S. aureus within1 hr and up to 24 hrs, preventing 100% of growth; these kinetic studiesindicated that half the MIC was bactericidal within 1 hr and alsoinhibited growth (50%) up to 24 hrs as compared to controls (FIG. 7)(CLSI. M100-S25 performance standards for antimicrobial susceptibilitytesting; Twenty-fifth informational supplement; 2015). Kinetic resultsdemonstrate the time efficacy of the conjugate against tested bacteriaand the sustained bactericidal activity over 24 hrs, supporting cleavageactivity in the presence of tested bacteria.

Next, we tested the conjugate against pre-formed bacterial biofilms ontwo different substrates (polystyrene and HA discs) to evaluateantimicrobial efficacy against biofilms for the first time in thiscontext, and to also determine if substrate specificity plays a role.Biofilms of S. aureus (ATCC-6538), and additionally biofilms ofPseudomonas aeruginosa (ATCC-15442), were subjected to BP-ciprofloxacinand antimicrobial activity was assessed. We also tested P. aeruginosahere because it is the second most common clinical pathogen inosteomyelitis, though far less frequent in prevalence than S. aureuscases. FIG. 8 shows results for polystyrene as the substrate for biofilmgrowth, and the minimal biofilm inhibitory concentration (MBIC⁵⁰) ofBP-ciprofloxacin was 15.6-31.2 mcg/mL for S. aureus ATCC-6538, which wascomparable to the MIC for this strain in planktonic cultures; no MBIC⁵⁰was observed for P. aeruginosa ATCC-15442 in the tested range ofconcentrations.

However, when HA discs were used as the biofilm substrate, markedlyimproved bactericidal activity was observed as shown in FIG. 9, and alltested concentrations of the conjugate resulted in statisticallysignificant bactericidal activity and reduction of colony forming units(CFUs). The MBIC⁵⁰ of the conjugate was 8 mcg/mL and the MBIC⁹⁰ was 50mcg/mL against S. aureus strain ATCC-6538; the MBIC⁹⁰ for the parentdrug ciprofloxacin was 8 mcg/mL against this pathogen. However, againstP. aeruginosa strain ATCC-15442 ciprofloxacin had no inhibitory orbactericidal activity while the conjugate was bactericidal in acidic andbasic conditions at 50 mcg/mL, and showed improved bactericidal activityin basic conditions as compared to S. aureus where improvedantimicrobial activity was observed in acidic conditions. Overall, theseresults suggest that the conjugate is more effective against biofilmpathogens in the presence of HA versus polystyrene as a substrate, andthat substrate specificity plays a role in antimicrobial activity inaddition to factors like strain of pathogen tested and mode of bacterialgrowth (planktonic versus biofilm). This has not been demonstratedpreviously and adds insight into antimicrobial potential of thesecompounds for clinical applications against biofilm pathogens.

Lastly, we performed antimicrobial tests with the conjugate in apreventative type of experimental setting with planktonic and biofilmcultures, which could also have clinical relevance in antibioticprophylactic scenarios for osteomyelitis. Here HA spherules wereintroduced to varying concentrations of BP-ciprofloxacin and theninoculated with S. aureus for 24 hrs, and quantitative assessmentsindicated no bacterial growth at concentrations as low as 7.8 mcg/mL andup to 250 mcg/mL of the conjugate, and minimal bacterial growth withstrong inhibition at conjugate concentrations ranging from 0.12 to 3.9mcg/mL as shown in FIG. 10.

We then used HA discs as substrates for growing S. aureus biofilmsagain, but this time the discs were rinsed with media after incubationof either BP-ciprofloxacin or ciprofloxacin prior to inoculation andbiofilm growth. FIG. 11 shows results of quantitative biofilm culturesand CFUs after 24 hrs of growth, and at 100 mcg/mL ciprofloxacininhibited all biofilm growth whereas at 10 mcg/mL BP-ciprofloxacininhibited all growth. Since the molecular mass of ciprofloxacin isapproximately half that of the conjugate, the conjugate was 20× moreactive in achieving complete bactericidal action as compared tociprofloxacin alone. These findings support an efficient mechanism ofenzymatic cleavage and release over time of the parent drugciprofloxacin from the prodrug. Efficient binding to HA and cleavage orregeneration of the parent antibiotic is requisite for conjugates inthis class to demonstrate substantial antimicrobial efficacy comparableor better than the parent antibiotic alone (Houghton, et al., Linkingbisphosphonates to the free amino groups in fluoroquinolones:preparation of osteotropic prodrugs for the prevention of osteomyelitis.J. Med. Chem 2008; 51:6955-69; (Tanaka, et al., Bisphosphonatedfluoroquinolone esters as osteotropic prodrugs for the prevention ofosteomyelitis. Bioorg Med Chem 2008; 16:9217-29; McPherson, et al.,Synthesis of osteotropic hydroxybiphosphonate derivatives offluoroquinolone anti bacterials).

In Vivo Safety and Efficacy:

Since this BP-ciprofloxacin conjugate is novel and has not been testedin vivo, we performed an initial safety and efficacy study in an animalmodel of peri-implant osteomyelitis. This model is a unique in-housejawbone peri-implant osteomyelitis model that was developed specificallyfor translational value to study biofilm-mediated disease and hostresponse in vivo (Freire, et al, Development of animal model forAggregatibacter actinomycetemcomitans biofilm-mediated oral osteolyticinfection: a preliminary study. J Periodontol 2011; 82:778-89). Briefly,biofilms of the jawbone osteomyelitis pathogen Aggregatibacteractinomycetemcomitans (Aa; wild-type rough strain D7S-1; serotype a),which is not indigenous to rat normal flora, are pre-inoculated onminiature titanium implants at 10⁹ CFU. To confirm Aa sensitivity to theparent drug ciprofloxacin prior to our animal studies, we performed ASTand MIC assays as performed for the long bone osteomyelitis pathogensdescribed previously. Disc diffusion inhibition zone assays revealeddiameters >40 mm, and the MIC⁹⁰ was 2 mcg/mL, indicating strongsusceptibility of this microbe to the parent drug ciprofloxacin. Aa hasalso been tested previously for susceptibility to a pH-sensitivebiotinylated ciprofloxacin prodrug and was found to be sensitive to theparent antibiotic (Manrique, et al., Perturbation of the indigenous ratoral microbiome by ciprofloxacin dosing. Mol Oral Microbiol 2013;28:404-14). After biofilms are established on the implants in vitro,they are surgically transferred to the jawbone of each rat. Animals areanesthetized, the cheeks are retracted and a transmucosal osteotomy isperformed so implants can be manually inserted into the osteotomy andsecured. Two biofilm-inoculated implants are placed in each rat (n=12rats and 24 implants) in the palatal bone bilaterally. This model allowsstandardized and reproducible quantities of viable bacteria to be formedas well-established biofilms on each implant, which we have previouslydemonstrated persists in vivo for several weeks after placement andcauses infection, inflammation and bone destruction locally (Freire, etal, Development of animal model for Aggregatibacteractinomycetemcomitans biofilm-mediated oral osteolytic infection: apreliminary study. J Periodontol 2011; 82:778-89).

Once peri-implant infection is established 1 week post-operatively, theanimals are dosed with BP-ciprofloxacin, ciprofloxacin alone as apositive control, and sterile endotoxin-free saline as a negativecontrol at the dosing regimens specified in the experimental section. Todetermine appropriate dosing concentrations, we calculated approximateinitial doses for the conjugate based on previous studies andpharmacokinetic data using similar target and release strategies andalso rodents (Houghton, et al., Linking bisphosphonates to the freeamino groups in fluoroquinolones: preparation of osteotropic prodrugsfor the prevention of osteomyelitis. J Med Chem 2008; 51:6955-69;Morioka, et al., Design, synthesis, and biological evaluation of novelestradiol-bisphosphonate conjugates as bone-specific estrogens. BioorgMed Chem 2010; 18:1143-8). We expected that increasing doses of 0.1, 1and 10 mg/kg BP-ciprofloxacin molar equivalents will allow us todetermine antimicrobial activity in 2 test animals per group based onsample size estimations and previous experience with the animal model(Freire, et al, Development of animal model for Aggregatibacteractinomycetemcomitans biofilm-mediated oral osteolytic infection: apreliminary study. J Periodontol 2011; 82:778-89). Animals were dosedvia intraperitoneal injection under general anesthesia, and allcompounds were constituted in sterile physiological injectable saline atappropriate pH. One week after pharmacotherapy, all animals weresacrificed and resection of peri-implant tissues was performed, andtissues were immediately homogenized and processed for quantitativeassessment of microbial load. Animals were monitored throughout thestudy period for local or systemic adverse effects of pharmacotherapy.

All animals tolerated the pharmacotherapy well with no cutaneousinjection-site reactions or inflammation, and no systemic adverse eventswere reported by managing veterinarians throughout the study period.Treatment efficacy was quantitatively measured in terms of the logarithmof the amount of viable bacteria (average log CFU per gram of tissue) asshown in FIG. 12.

In vivo, the animals dosed with the conjugate at 0.3 mg/kg in multipledoses (×3) over the course of a week demonstrated no recovery of Aa or100% killing. A single dose of BP-ciprofloxacin at 10 mg/kg also showedhigh efficacy with 2 log reduction or 99% bacterial killing and morethan an order of magnitude greater activity than ciprofloxacin alone atthe same total concentration but in multiple doses. Ciprofloxacin alonein a multiple dosing regimen resulted in 1 log reduction or 90%bacterial killing, which was expected and why we chose it as thepositive control given the known efficacy of this compound, itsantimicrobial activity, and the fact that it represents the parent drugof the conjugate. Conjugate concentrations of 0.1 and 1 mg/kg had littleeffect, suggesting that further optimization is possible in thiscontext. Nonetheless, given the targeting and release ability of theprodrug, effective doses can be reasonably achieved in a clinicalsetting given the safety profile of constituent compounds and theability to dose orally or intravenously. Interestingly, in the conjugatemultiple dosing group our cultures showed evidence of yeast morphologyand no recoverable Aa. One explanation for this phenomenon could becontamination, although this is highly unlikely since methodology wasperformed similarly and simultaneously, yeast is not cultured in ourlaboratory, the only animal samples where Aa was not recovered were inthis same multiple dosing group and in two separate animals. Therefore,a more likely explanation is that killing and resolution of Aa occurredin vivo and that another organism less sensitive to the parent drugciprofloxacin grew in our cultures, such as yeast. In fact rats are usedas a well-established model for oral candidiasis and their equivalent tonormal human oral flora yeast is Candida pintolopessi, which can causeunexpected disease in antibiotic-treated or immune-compromised rodents(Junqueira. Models hosts for the study of oral candidiasis. Adv Exp MedBiol. 2012; 710:95-105). This is also a well-known phenomenon in humanpatients treated with antibiotics, namely yeast overgrowth orcandidiasis due to suppression of bacterial flora that normally competeswith yeast in vivo.

The resolution of infection over time in vivo with the conjugate ascompared to negative controls, and also as compared to the positivecontrol parent drug, further supports that the conjugate bindseffectively to bone and releases the parent antibacterial agent. Lack ofefficacy in this model would suggest either that the prodrug is notbinding to or that it is not releasing the parent drug. This provides atleast an indirect way to understand the pharmacokinetics of the prodrugin vivo (Houghton, et al., Linking bisphosphonates to the free aminogroups in fluoroquinolones: preparation of osteotropic prodrugs for theprevention of osteomyelitis. J. Med. Chem. 2008; 51:6955-69). A similarstudy but in a rat tibia osteomyelitis model tested the activity ofBP-fluoroquinolones and found similar efficacy and evidence of greatlyenhanced antimicrobial activity of tested conjugates, but in apreventative context where a single intravenous injection of the prodrugwas administered 1-2 days before an infection of the bone (Houghton, etal., Linking bisphosphonates to the free amino groups influoroquinolones: preparation of osteotropic prodrugs for the preventionof osteomyelitis. J. Med. Chem. 2008; 51:6955-69). The infection in thismodel was created by injecting a bolus of planktonic bacteria in thesurgically exposed tibia and the animals were sacrificed 24 h afterinfection. This study was not a biofilm-mediated osteomyelitis treatmentstudy, but is consistent with in vitro data presented hereindemonstrating that biofilm growth can be prevented with pre-treatment ofBP-ciprofloxacin (Houghton, et al., Linking bisphosphonates to the freeamino groups in fluoroquinolones: preparation of osteotropic prodrugsfor the prevention of osteomyelitis. J. Med. Chem. 2008; 51:6955-69).Our experiment confirms the ability of a BP-ciprofloxacin prodrug atsafe and adequate single dose produces a sufficient concentration of theparent drug to maintain bactericidal activity against establishedbiofilms when the activity of the parent antibiotic alone has alreadydiminished. This conjugate will be further evaluated for the ability totreat long bone osteomyelitis in an animal model, and comprehensivepharmacokinetic and pharmacodynamic studies will also be performed invivo; the results of these examinations will be presented in due course.

Discussion

This Example demonstrates successful design and synthesis of a phenylcarbamate BP-ciprofloxacin conjugate utilizing a target and releasestrategy, and systematically evaluated functionality of each constituentof this compound (as well as the conjugate as a whole) in vitro and invivo. In vitro antimicrobial investigations of BP-ciprofloxacin testedagainst common osteomyelitis pathogens revealed a strong bactericidalprofile, and safety and efficacy was demonstrated in vivo in an animalmodel of peri-prosthetic osteomyelitis. In vivo, the animals dosed withthe conjugate at 0.3 mg/kg in multiple doses (0.9 mg/kg total) over thecourse of a week demonstrated optimal efficacy with no recoverablebacteria. A single dose of 10 mg/kg of conjugate (5 mg ciprofloxacinconsidering the molecular mass of the conjugate is twice that of theparent drug) also showed strong antimicrobial activity and resulted in99% killing of bacteria. The multiple dosing of the conjugate and thehighest single dose of the conjugate were superior to multiple dosing ofthe parent antibiotic ciprofloxacin at 30 mg/kg. Lower single doseconcentrations (0.1 and 1 mg/kg) of the conjugate were not efficacious.

These findings indicate a minimum dose is necessary for in vivo efficacyof the conjugate when given as a single dose, but that a much lowerconcentration of the conjugate when dosed regularly can provide greatestefficacy and at < 1/10^(th) the concentration of the parent antibiotic.For translation to practice this targeting strategy could prove usefulby reducing dosing concentrations for patients and improving therapeuticindex, and also by limiting systemic exposure. Importantly, theseresults along with other studies in this field are indicating thatdirect comparisons between these prodrugs and their parent compound aresomewhat arbitrary as conjugates have unique pharmacometric parameters.Any future pharmacokinetic modeling for conjugates in this class wouldhave to include a skeletal compartment of distribution mathematically,which is not generally done with antibiotic pharmacokinetic studies.This would provide for novel pharmacological data and also has in vivoimplications.

BP-ciprofloxacin was also tested against clinically relevant biofilmsfor the first time here, and demonstrated strong antimicrobial activitywhen biofilms were attached to bone as a substrate both in vitro and invivo. Antimicrobial activity of the conjugate appears to be associatedwith many parameters, including the species and strain of pathogentested, its mode of growth (biofilm versus planktonic), substrate forbiofilm colonization, pH, concentration, bone binding affinity andrelease kinetics. Optimization of this class of conjugates using BPs asbiochemical vectors for the delivery of antimicrobial agents to bone(where biofilm pathogens reside) through a target and release strategyshould represent an advantageous approach to the treatment ofosteomyelitis and provide for improved pharmacokinetics while minimizingsystemic toxicity.

Materials and Methods

Chemistry:

1-(benzyloxy)-4-(bromomethyl)benzene (1)

4-Benzyloxy benzyl alcohol (1.00 g, 4.67 mmol) was dissolved inanhydrous diethyl ether (25 ml) in an oven-dried flask under nitrogen.The flask was cooled in an ice bath. Bromotrimethylsilane (BTMS, 1.26ml, 9.52 mmol) was added by syringe. The flask was allowed to slowlywarm to room temperature. After 17 h of stirring, the reaction mixturewas poured into water (50 ml) and the organic phase was separated. Theaqueous phase was washed with diethyl ether (2×20 ml) then the combinedorganic phase was washed with brine (2×20 ml) and dried over sodiumsulfate. Evaporation of the ether gave the product as a whitecrystalline solid (1.23 g, 95% yield) ¹H NMR (400 MHz, Chloroform-d) δ7.47-7.28 (m, 7H), 6.98-6.90 (m, 2H), 5.07 (s, 2H), 4.50 (s, 2H).

Tetraisopropyl (2-(4-(benzyloxy)phenyl)ethane-1,1-diyl)bis(phosphonate)(2)

Under nitrogen protection, anhydrous THF (2 ml) was added to sodiumhydride 57-63% dispersion in mineral oil. Tetraisopropyl methylenediphosphonate (0.57 ml, 1.8 mmol) was added dropwise with stirring atroom temperature. Gas was evolved and the grey suspended solid wasconsumed leaving a mostly clear solution. The mixture was stirred afurther 10 min. Solid 1 was added in one portion under nitrogencounterflow. Solution remained clear for 1 min and then became cloudy.Stirring was maintained for 2 h then the reaction was checked by TLC(100% EtOAc visualized by UV or cerium ammonium molybdate (CAM) stain)two new spots were apparent at RF=0.37 and 0.58. Some 1 (RF>0.9)remained, reaction was heated to 50° C. for 30 min, little progress wasapparent by TLC) Reaction mixture was poured into 5% aqueous citric acidand extracted with ether (2×30 ml), washed with brine and evaporated.The residue was purified by flash chromatography using 230-400 meshsilica using 10% EtOAc in hexane increasing to 100% EtOAc as eluent.Desired compound was obtained as a colorless oil (0.508 g, 52% yield)

Tetraisopropyl (2-(4-hydroxyphenyl)ethane-1,1-diyl)bis(phosphonate) (3)

Compound 2 (0.508 g, 0.925 mmol) was dissolved in 13 ml of methanol and70 mg of 10% palladium on carbon was added. The flask was flushed withnitrogen, then hydrogen and stirred overnight with a hydrogen balloon inplace. TLC (10% MeOH in EtOAc, vis. w/ UV or CAM stain) showeddisappearance of the starting material (RF=0.63) and appearance of a newspot with RF=0.49. The reaction mix was filtered through celite with 100ml of methanol. Evaporation of the filtrate gave the desired compound asa slightly yellow oil (0.368 g, 88% yield) that was used without furtherpurification. ¹H NMR (400 MHz, Chloroform-d) δ 7.07 (d, J=8.2 Hz, 2H),6.69 (d, J=8.2 Hz, 2H), 4.71 (m, 4H), 3.11 (td, J=16.9, 6.0 Hz, 2H),2.47 (tt, J=24.4, 6.0 Hz, 1H), 1.32-1.21 (m, 24H). 31P NMR (162 MHz,Chloroform-d) δ 21.06.

4-(2,2-bis(diisopropoxyphosphoryl)ethyl)phenyl (4-nitrophenyl) carbonate(4)

Compound 3 (0.171 g, 0.380 mmol) was dissolved in 8 ml ofdichloromethane then triethylamine (159 μl, 1.14 mmol) was addedfollowed by p-nitrophenyl chloroformate (0.086 g, 0.418 mmol) in oneportion. The solution turned from colorless to yellow immediately. Afterstirring for 2.5 h, TLC (5% MeOH in EtOAc, UV visualization) showed onlya trace of starting material (RF=0.31) and appearance of a strong spotat RF=0.59. The compound was purified by flash chromatography using 1:1ethyl acetate:hexane as eluent to remove one impurity (RF=0.88) beforeeluting the product with pure ethyl acetate. ¹H NMR (400 MHz,Chloroform-d) δ 8.29 (d, J=9.1 Hz, 2H), 7.46 (d, J=9.1 Hz, 2H), 7.33 (d,J=8.5 Hz, 2H), 7.15 (d, J=8.6 Hz, 2H), 4.84-4.58 (m, 4H), 3.22 (td,J=16.5, 6.2 Hz, 2H), 2.47 (tt, J=24.1, 6.2 Hz, 1H), 1.33-1.14 (m, 24H).

7-(4-((4-(2,2-bis(diisopropoxyphosphoryl)ethyl)phenoxy)carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (5)

Ciprofloxacin (46.5 mg, 0.140 mmol) was suspended in 1.4 ml of water ina plastic vial. 151 μl of 1 M HCl was added and the vial was vortexed todissolve ciprofloxacin giving a clear colorless solution. Na₂CO₃ wasadded to adjust the pH to 8.5 and a thick white precipitate formed. Thevial was placed in an ice bath and Compound 4 (71.9 mg, 0.117 mmol)dissolved in 1.4 ml of THF was added dropwise over about 5 min. The vialwas then removed from the ice bath, protected from light and stirredovernight at room temperature. The reaction mixture turned bright yellowwith suspended solid. TLC (5% MeOH in EtOAc) showed disappearance of thestarting material 4 and appearance of a fluorescent blue spot (RF=0.51)and a visible yellow spot (RF=0.816) attributed to p-nitro phenolbyproduct. The reaction mixture was diluted with 10 ml of water andfiltered through a fine glass frit. The retained solid was washed withwater until no yellow color remained. The solids were then dissolved andwashed from the frit with DCM and the solution was loaded onto a flashsilica column and eluted with DCM increasing MeOH concentration to 5% toelute a band with light blue fluorescence. Combined fractions wereevaporated to give the title compound as a white solid. ¹H NMR (400 MHz,Methanol-d4) ¹H NMR (400 MHz, Methanol-d4) δ 8.79 (s, 1H), 7.93 (d,J=13.3 Hz, 1H), 7.54 (s, 1H), 7.30 (d, J=8.4 Hz, 2H), 7.05 (d, J=8.5 Hz,2H), 4.70 (dpd, J=7.4, 6.2, 1.3 Hz, 4H), 3.90 (s, 5H), 3.75 (s, 3H),3.39 (s, 4H), 3.18 (td, J=16.6, 6.4 Hz, 2H), 2.65 (tt, J=24.3, 6.3 Hz,1H), 1.43-1.34 (m, 1H), 1.34-1.19 (m, 24H), 1.18-1.10 (m, 2H).

1-cyclopropyl-7-(4-((4-(2,2-diphosphonoethyl)phenoxy)carbonyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (6), Also Referred to Herein as Formula 2

Compound 5 (10.0 mg, 0.0124 mmol) was dissolved in DCM (0.2 ml) in a 1.5ml vial and bromotrimethylsilane (BTMS) (0.2 ml) was added and the vialwas quickly capped and immersed in a 35° C. oil bath. After stirring for24 h, solvent and BTMS were removed by evaporation and 1 ml of MeOH wasadded and the vial stirred overnight. Evaporation of solvent left 6.82mg (0.107 mmol, of pale yellow solid with green fluorescence. A sample˜0.2 mg was taken for HPLC analysis. Suspended in water, pH was measuredat 2.5 then adjusted to 6.7 to give a slightly yellow solution with bluefluorescence. HPLC analysis (Luna C₁₈, buffer system 0.1 M NH₄OAc bufferpH 7.1; A: 20% acetonitrile, B: 70% acetonitrile. 0-7 min: 100% A, 7-25min gradient 0-100% B) showed major peak at RT=14.8 min and minor peaksat 5.76 min (assigned to ciprofloxacin) and 18.8 min. Ciprofloxacinstandard (saturated solution in buffer A diluted 2×, 5 μl injection)gave an RT of 5.68 min.

Microbiology:

Experimental strains: Twelve S. aureus clinical osteomyelitis strains ofmethicillin-susceptible profile and one clinical methicillin-resistantstrain (MR-CIPS) were tested. These pathogens are part of the straincollection of the Department of Pharmaceutical Microbiology andParasitology Wroclaw Medical University, Poland. Additionally, thefollowing ATCC collection strains were chosen for experimental purposes:S. aureus 6538 and P. aeruginosa 15442.

HA discs: For custom disc manufacturing, commercially available HApowder was used. Powder pellets of 9.6 mm in diameter were pressedwithout a binder. Sintering was performed at 900° C. The tablets werecompressed using the Universal Testing System for static tensile,compression, and bending tests (Instron model 3384; Instron, Norwood,Mass.). The quality of the manufactured HA discs was checked by means ofconfocal microscopy and microcomputed tomography (micro-CT) using anLEXT OLS4000 microscope (Olympus, Center Valley, Pa.) and Metrotom 1500microtomograph (Carl Zeiss, Oberkochen, Germany), respectively.

Disc diffusion test to evaluate sensitivity of tested strains tociprofloxacin: This procedure was performed according to EUCASTguidelines. Briefly, 0.5 McFarland (MF) of bacterial dilution was spreadon Mueller-Hinton (MH) agar plate. The discs containing 5 mg ofciprofloxacin were introduced and the plate was subjected to incubationat 37° C./24 h. Next, inhibition zones were recorded using a ruler.Obtained values (mm) were compared to appropriate values of inhibitionzone from EUCAST tables.

Evaluation of MIC of tested compounds against planktonic forms ofclinical staphylococcal strains analyzed: To assess the impact ofBP-ciprofloxacin and ciprofloxacin on microbial growth, 100 μl ofmicrobial solutions of density of 1×10⁵ cfu/ml were placed into wells of96-well test plate together with appropriate concentrations of testedcompounds. Immediately after that, the absorbance of solutions wasmeasured using a spectrometer (Thermo Scientific Multiscan GO) at 580 nmwavelength. Subsequently, the plate was incubated for 24 h/37° C. in ashaker to obtain optimal conditions for microbial growth and to preventbacteria from forming biofilms. After incubation, the absorbance wasmeasured once again. The following control samples were established:negative control sample one: sterile medium without microbes; negativecontrol sample two: sterile medium without microbes implemented withDMSO (dimethyl sulfoxide, Sigma-Aldritch) to final concentration of 1%(v/v); positive control sample one: medium+microbes with no compoundtested; positive control sample two: medium+microbes with no compoundtested but implemented with DMSO to final concentration of 1% (v/v).Rationale for use of 1% DMSO was that ciprofloxacin dissolvesefficiently in this solvent, however, concentrations of DMSO>1% might bedetrimental for microbial cells. To assess relative number of cells, thefollowing calculations were performed. The value of absorbance ofcontrol samples (medium+microbes in case of BP-ciprofloxacin,medium+microbes+DMSO for ciprofloxacin) was estimated at 100%. Next, therelative number of cells subjected to incubation with tested compoundswere counted as follows: value of control sample absorbance/value oftested sample*100%.

Spectroscopic analysis of BP-ciprofloxacin conjugate in trypticase soybroth (TSB) microbiological media to test stability: BP-ciprofloxacin infinal concentrations of 0.24-250 mg/L in TSB microbiological medium wasintroduced to wells of 96-well plate. Immediately afterwards theabsorbance of solutions was measured using a spectrometer (ThermoScientific Multiscan GO) at 275 nm wavelength. Next, solutions were leftfor 24 h/37° C./shaking. After incubation, absorbance was measured onceagain. To assess for degradation of conjugate, values of absorbancetaken at 0 hr and 24 hrs were compared.

Spectroscopic analysis of BP-ciprofloxacin conjugate in trypticase soybroth microbiological media with the addition of HA spherules: VariousBP-ciprofloxacin concentrations were introduced to HA powder (spherules)suspended in TSB microbiological medium. Solutions containingBP-ciprofloxacin and HA spherules were introduced to wells of 24-wellplate. Final concentration of powder was 10 mg/1 mL, while finalconcentration of conjugate was 0.24-250 mg/L. Immediately afterwards theabsorbance of solutions was measured using a spectrometer (ThermoScientific Multiscan GO) at 275 nm wavelength. Plates were shakenautomatically in the spectrometer prior to assessment. Next, plates wereleft for 24 h/37° C./shaking. After 24 hours, absorbance was measuredonce again. To assess the relative concentration of the conjugate at 0hr and 24 hrs, values of absorbance taken in the beginning and at theend of experiment were compared.

Antimicrobial susceptibility testing of BP-ciprofloxacin againstplanktonic cultures of S. aureus strain ATCC-6538 in acidic versus basicpH: This experimental setting was performed in the same manner asdescribed previously for disc diffusion testing, but microbiologicalmedia was adjusted to pH 7.4 and pH 5 using KOH or HCL solution andmeasured using a universal pH-indicator (Merck, Poland).

Time-kill assay for BP-ciprofloxacin conjugate against S. aureus strainATCC-6538 (MSSA) and clinical MRSA strain MR4-CIPS: This experiment wasperformed in the same manner as described previously under thesubheading: “Evaluation of MIC of tested compounds against planktonicforms of clinical staphylococcal strains analyzed”, but absorbanceassays (at 580 nm wavelength) were taken in hour: 0, 1, 2, 4, 8, 16, 24.

Antimicrobial susceptibility testing of BP-ciprofloxacin againstpreformed biofilms of S. aureus strain ATCC-6538 and P. aeruginosastrain ATCC-15442: Strains cultured on appropriate agar plates (Columbiaagar plate for S. aureus; MacConkey agar plate for P. aeruginosa) weretransferred to liquid microbiological media and incubated for 24 h/37°C. under aerobic conditions. After incubation, strains were diluted tothe density of 1 MF. The microbial dilutions were introduced to wells of24-well plates containing HA discs as a substrate, or simply topolystyrene wells where the bottom surface of the wells served as thesubstrate for biofilm development. Strains were incubated at 37° C. for4 hrs. Next, the microbe-containing solutions were removed from thewells. The surfaces, HA discs and polystyrene plates, were gently rinsedto leave adhered cells and to remove planktonic or loosely-boundmicrobes. Surfaces prepared in this manner were immersed in fresh TSBmedium containing 0.24-125 mg/L of BP-ciprofloxacin conjugate. After 24hrs of incubation at 37° C. the surfaces were rinsed using physiologicalsaline solution and transferred to 1 mL of 0.5% saponin (Sigma-Aldrich,St Louis, Mo.). The surfaces were vortex-mixed vigorously for 1 minuteto detach cells. Subsequently, all microbial suspensions were diluted 10to 10⁹ times. Each dilution (100 mL) was cultured on the appropriatestable medium (MacConkey, Columbia for P. aeruginosa and S. aureus,respectively) and incubated at 37° C. for 24 hours. After this time, themicrobial colonies were counted and the number of cells forming biofilmwas assessed. Results were presented as the mean number of CFU persquare millimeter surface ±standard error of the mean. To estimate theexact surface area of HA discs, x-ray tomographic analysis was applied.For estimation of the area of test plate bottoms, the equation forcircle area: πr2 was applied.

Preventative ability of BP-ciprofloxacin conjugate to inhibit S. aureus6538 adherence to HA spherules: Various BP-ciprofloxacin concentrationswere introduced to HA powder (spherules) suspended in TSBmicrobiological medium. Solutions containing conjugate and HA spheruleswere introduced to wells of 24-well plates. Final concentrations ofpowder were 10 mg/1 mL, while final concentrations of the conjugate were0.12-250 mg/L. Suspensions were left for 24 h/37° C./shaking. After 24h, suspensions were removed from the wells and impulse-centrifuged toprecipitate HA powder. Next, supernatant was very gently discarded and afresh 1 mL of S. aureus of density 10⁵ cfu/mL was introduced to the HAspherules. Subsequently, this solution was shaken, absorbance wasmeasured using 580 nm wavelength and left for 24 h/37° C./shaking. Afterincubation absorbance was measured again and values from 0 hr and 24 hrswere compared to assess reduction of bacterial growth with regard tocontrol sample one (bacterial suspension but no spherules) and controlsample two (bacterial suspension+spherules but with no conjugate added).Additionally, solutions were impulse-centrifuged, supernatant was gentlydiscarded, while bacteria-containing HA spherules were culture plated asbefore and quantitatively assessed.

Survival of S. aureus after 24 hrs of incubation in presence ofconjugate-coated HA discs: HA discs were immersed in 2 mL of solutioncontaining various concentrations of BP-ciprofloxacin or ciprofloxacinalone and left for 24 h/37° C. HA discs incubated in DMSO or phosphatebuffer served as control samples. Next, discs were rinsed 3 times withsterile water. After rinsing, 2 mL of 0.5 MF of. S. aureus ATCC6538 wereintroduced to wells containing HA discs as a substrate for biofilmdevelopment and biofilms were formed as before.

Animal Study:

All animal protocols and procedures were approved and performed inaccordance with the Institutional Animal Care and Use Committee (IACUC)of the University of Southern California (USC), and in accordance withthe Panel on Euthanasia of the American Veterinary Medical Association.USC is registered with the United States Department of Agriculture(USDA), has a fully approved Letter of Assurance (#A3518-01) on filewith the National Institutes of Health (NIH) and is accredited by theAmerican Association for the Accreditation of Laboratory Animal Care(AAALAC). USC's animal welfare assurance number is A3518-01. The titleof our IACUC approved protocol is: “Bone targeted antimicrobials forbiofilm-mediated osteolytic infection treatment”, and the protocolnumber is 20474. For this study 12 five-month-old, virgin, femaleSprague-Dawley rats weighing approximately 200 g each were used in thisstudy. Two animals were housed per cage in a vivarium at 22° C. under a12-h light/12-h dark cycle and fed ad libitum with a soft diet (PurinaLaboratory Rodent Chow). All animals were treated according to theguidelines and regulations for the use and care of animals at USC.Animals were under the supervision of full-time veterinarians on call 24hrs/day who evaluate the animals personally on a daily basis. All animalexperiments are described using the ARRIVE guidelines for reporting onanimal research to ensure the quality, reliability, validity andreproducibility of results (Kilkenny, et al., Improving bioscienceresearch reporting: the ARRIVE guidelines for reporting animal research.Vet Clin Pathol 2012; 41:27-31).

This animal model is an in-house jawbone peri-implant osteomyelitismodel designed specifically to study biofilm-mediated disease and hostresponse in vivo (Freire, et al, Development of animal model forAggregatibacter actinomycetemcomitans biofilm-mediated oral osteolyticinfection: a preliminary study. J Periodontol 2011; 82:778-89). Biofilmsof the jawbone osteomyelitis pathogen Aa were pre-formed on miniaturetitanium implants at 10⁹ CFU. To confirm Aa sensitivity to the parentdrug ciprofloxacin prior to our animal studies, we performed AST and MICassays as performed for the long bone osteomyelitis pathogens describedpreviously. After biofilms were established on the implants in vitro,they were surgically transferred to the jawbone of each rat. Forsurgery, animals were anesthetized with 4% isoflurane inhalant initiallyfollowed by intraperitoneal injection of ketamine (80-90 mg/kg) plusxylazine (5-10 mg/kg). Then local anesthesia was given via infiltrationinjection of bupivicaine 0.25% at the surgical site. Buprenorphinesustained release (1.0-1.2 mg/kg) was then given subcutaneously aspreemptive analgesia before making initial incisions. Once anesthetized,the buccal mucosa of each rat was retracted and a transmucosal osteotomywas performed with a pilot drill into the alveolar ridge in the naturaldiastema of the anterior palate. Implants were then manually insertedinto the osteotomy and secured into the bone until the platform is atmucosal level. Two biofilm-inoculated implants were placed in each ratin the palatal bone bilaterally.

One week post-operatively isoflurane 4% was given again to brieflyanesthetize the rats and check implant stability and document clinicalfindings at the implant and infection site. The animals were then dosedvia intraperitoneal injection with BP-ciprofloxacin (0.1 mg/kg, 1 mg/kg,or 10 mg/kg as a single dose, and at 0.3 mg/kg 3×/week for a multipledosing group) or ciprofloxacin alone (10 mg/kg 3×/week also as amultiple dosing group) as a positive control, and sterile endotoxin-freesaline as a negative control. Allocation of animals to treatment andcontrol groups was done through a randomization process. The multipledosing group animals were anesthetized as before prior to eachadditional injection over the course of the week. All compounds werepharmacological grade and constituted in sterile physiologicalinjectable saline at appropriate pH. One week after pharmacotherapy, allanimals were euthanized in a CO₂ chamber (60-70% concentration) for fiveminutes, followed by cervical dislocation. Resection of peri-implanttissues (1 cm²) was performed en bloc and implants were removed.Peri-implant tissues were immediately homogenized and processed forquantitative assessment of microbial load. Rat allocations to treatmentand control groups were de-identified and concealed from subsequentinvestigators analyzing the microbial data. For microbial analysis,peri-implant soft tissue and bone was processed by placement in 1 mL of0.5% saponine and vortexed for 1 minute before being transferreddirectly to agar plates and cultured. The medium for culturing Aaconsisted of modified TSB and frozen stocks were maintained at −80° C.in 20% glycerol, 80% modified TSB. All culturing was performed at 37° C.in 5% CO₂. The numbers of CFU in the homogenate (numbers of CFU pergram) was determined by plating aliquots of the serially dilutedhomogenate onto TSA plates. The reduction in the mean log 10 number ofCFU per gram as a function of treatment was recorded.

Statistical Analysis:

Statistical calculations were performed with the SigmaStat package,version 2.0 (SPSS, Chicago, Ill.). Power analyses were performed todetermine sample size estimation for in vitro and in vivo studies priorto experimentation using G Power 3 software (Faul F, Erdfelder E,Buchner A, Lang A G. Statistical power analyses using G*Power 3.1: testsfor correlation and regression analyses. Behav Res Meth 2009;41:1149-60). Quantitative data from experimental results was analyzedusing the Kruskall-Wallis test or one-way ANOVA and statisticalsignificance was accepted at p<0.05 when comparing treatments tocontrols.

Example 2

methyl 4-(2,2-bis(diisopropoxyphosphoryl)ethyl)benzoate (7)

Under nitrogen atmosphere, in a 50 mL 2-neck round bottom flask, THF (3mL) was added to 60% dispersion of NaH in mineral oil (0.122 g, 3.05mmol). The suspension was cooled to 0° C., while stirring, andtetraisopropyl methylenediphosphonate (0.69 mL, 2.18 mmol) was addedgradually. The reaction was allowed to reach ambient temperature andonce hydrogen gas stopped bubbling out of the reaction mixture, thesolution was cooled to 0° C. again. Methyl 4-(bromomethyl)benzoate (0.5g, 2.18 mmol) was dissolved in THF (2 mL) and added to the reactiondropwise. The resulting solution was allowed to stir overnight whileslowly reaching ambient temperature. Reaction mixture was then cooled to0° C. and quenched with H₂O (1 mL). 5% aqueous solution of citric acidin water (30 mL) and extracted with Et₂O (3×30 mL) combined organicswere washed with brine (50 mL), dried on Na₂SO₄, filtered, concentratedunder reduced pressure and purified by silica gel column chromatographyusing a EtOAc:Hex gradient (10-100%) to afford 7 as a faint yellow oil(0.323 g, 30% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.93 (d, J=8.0 Hz, 2H),7.33 (d, J=8.4, 6.0 Hz, 2H), 4.79-4.683 (m, 4H), 3.88 (s, 3H), 3.24 (td,J=16.0, 6.4 Hz, 2H), 2.50 (tt, J=24.0, 6.2 Hz, 1H), 1.34-1.24 (m, 24H).³¹P NMR (162 MHz, Chloroform-d) δ 20.57.

4-(2,2-bis(diisopropoxyphosphoryl)ethyl)benzoic Acid (8)

To a solution of 7 (0.278 g, 0.583 mmol) in MeOH (3 mL) in a 8 Dr glassvial, LiOH.H₂O (0.122 g, 2.914 mmol) was added and the resultingsolution was stirred at room temperature overnight. The reaction mixturewas evaporated to dryness, the residue was dissolved in water (30 mL),and HCl_((aq)) (1 M) was added slowly to reach pH 3. The resultingmixture was extracted with CHCl₃ (3×30 mL). Combined organics were driedon MgSO₄ and concentrated under reduced pressure to afford a thick clearoil. Yield: quantitative. ¹H NMR (400 MHz, CDCl₃): δ=7.96 (d, J 6.4,2H), 7.36 (d, J 6.4, 2H), 4.78 (sex, J 5.0, 4H), 3.27 (td, J 14.0, 4.8,2H), 2.60 (tt, J 20.0, 4.8, 1H), 1.43-1.26 (m, 24H). ³¹P NMR (162 MHz,Chloroform-d) δ 20.57.

tetraisopropyl(2-(4-(chlorocarbonyl)phenyl)ethane-1,1-diyl)bis(phosphonate) (9)

Under nitrogen atmosphere, Compound 8 (0.162 g, 0.339 mmol) wasdissolved in chloroform (1 ml) and catalytic amount of DMF (1.3 μL,0.017 mmol) was added. Thionyl chloride (49.2 μL, 0.678 mmol) was addedslowly and the reaction was allowed to stir for 2 hours at roomtemperature. Solvents were removed under vacuum to afford a clear oil.The product was immediately used in the next step without furthermanipulation. Yield: quantitative.

7-(4-(4-(2,2-bis(diisopropoxyphosphoryl)ethyl)benzoyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate(10)

Ciprofloxacin (0.112 g, 0.339 mmol) was suspended in chloroform (1 ml)and N,N-diisopropylethylamine (DIPEA) (354.3 μL, 2.034 mmol) was added.Freshly made compound 9 was dissolved in chloroform (1 mL) and graduallyadded to the Ciprofloxacin:DIPEA suspension. Reaction mixture wascovered with foil and allowed to stir at room temperature overnight. Thefollowing day, solvents were removed under vacuum and the resultingcrude was dissolved in DCM (5 mL) and filtered through a medium fritfunnel and washed with more DCM (3×5 mL). The filtrate was concentratedunder vacuum and further purified by silica gel column chromatographyusing a MeOH:DCM gradient (0-10%) to afford 10 as a viscous oil thatgradually solidified (0.226 g, 84% yield, 1.8 eq DIPEA salt). ¹H NMR(400 MHz, CDCl₃) δ=8.79 (s, 1H), 8.06 (d, J 12.8, 1H), 7.38 (m, 5H),4.80-4.73 (m, 4H), 4.00 (s, br, 4H), 3.56-3.53 (m, 1H), 3.33-3.20 (m,6H) 2.50 (m, 1H), 1.45-1.38 (m, 2H), 1.32-1.25 (m, 24H), 1.23-1.19 (m,2H). ³¹P NMR (162 MHz, Chloroform-d) δ 20.77.

1-cyclopropyl-7-(4-(4-(2,2-diphosphonoethyl)benzoyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (11)

In a 8 Dr glass vial, compound 10 (0.108 g, 0.136 mmol) was dissolved inDCM (700 μL) and BTMS (686.0 μL, 5.200 mmol) was added. The vial wascapped and heated overnight at 35° C. while covered with foil andstirring. The following day, solvent was removed under vacuum and thecrude was quenched with MeOH (2 mL). The resulting solution was stirredat room temperature for 30 minutes. Solvent was removed under vacuum toafford an orange oil. A few drops of water was added to ppt a yellowsolid. More MeOH (2 mL) was added and the resulting suspension wasfiltered using a medium fritted glass funnel. The resulting solid wasfurther washed with MeOH to afford a yellow powder (0.070 g, 82% yield).¹H NMR (400 MHz, D₂O, pH 7.5): δ=8.54 (s, br, 1H), 7.89 (m, 1H), 7.64(m, 1H), 7.54 (d, J 8.0, 2H), 7.44 (d, J 8.0, 2H), 4.79 (m, overlap withD₂O, 4H), 4.00 (s, br, 2H), 3.79 (s, br, 2H), 3.47 (s, br, 2H), 3.34 (s,br, 2H), 3.21 (td, J 14.0, 6.4, 2H), 2.30 (tt, J 22.0, 6.6, 1H). ³¹P NMR(162 MHz, Chloroform-d) δ 19.12. ESI-MS m/z (−): 622.24 [M−H].

Example 3

Non-limiting examples of quinolones that can be included in the BPconjugates.

Fluorinated Quinolones

The following is an example of a non-flouronated quinolone.

Example 4

The following are non-limiting examples of BP-quinolone conjugates asdescribed herein.

Example 5

Infectious bone disease, or osteomyelitis, is a major problem worldwidein human¹ and veterinary² medicine and can be devastating due to thepotential for limb-threatening sequelae³ and mortality.⁴ The currentapproach to treat osteomyelitis is mainly antimicrobial, and oftenintravenous and long-term, with surgical intervention in many cases tocontrol infection. The causative pathogens in the majority of long boneosteomyelitis cases are biofilms of Staphylococcus aureus; thesemicrobes are bound to bone (FIG. 1) in contrast to their planktonic(free-floating) counterparts.⁵

The biofilm-mediated nature of osteomyelitis is important in clinicaland experimental settings because many biofilm pathogens areuncultivable and exhibit an altered phenotype with respect to growthrate and antimicrobial resistance.^(5, 6) The difficulty in eradicatingbiofilms with conventional antibiotics partly explains why the highsuccess rates of antimicrobial therapy in general have not yet beenrealized for orthopedic infections, along with the development ofresistant biofilm pathogens, poor penetration of antimicrobial agentsinto bone, and adverse events related to systemic toxicity.³

To overcome the many challenges associated with osteomyelitistreatment,⁷ there is increasing interest in drug delivery approachesusing bone-targeting conjugates to achieve higher or more sustainedlocal therapeutic concentrations of antibiotic in bone while minimizingsystemic exposure.⁸ Conjugation of fluoroquinolone antibiotics toosteoadsorptive bisphosphonates (BPs) (FIG. 13) represents a promisingapproach because of the long clinical track-record of safety of eachconstituent, and their advantageous biochemical properties.^(9, 10)Ciprofloxacin (FIG. 13) has several advantages for repurposing in thiscontext: 1) it can be administered orally or intravenously with relativebioequivalence, 2) it is already FDA approved and indicated for bone andjoint infections caused by Pseudomonas aeruginosa and several otherpathogens, 3) it has broad spectrum antimicrobial activity that includesthe most commonly encountered osteomyelitis pathogens likeStaphylococcus aureus (methicillin-susceptible), Pseudomonas aeruginosafor long bone osteomyelitis,¹¹ and Aggregatibacter actinomycetemcomitansfor jawbone osteomyelitis,¹² ⁴) it demonstrates bactericidal activity inclinically achievable doses,^(13 and 5)) it is the least expensive drugin the fluoroquinolone family.

However, like most antibiotics, fluoroquinolones suffer from reducedactivity against biofilms as compared to the same bacteria in planktonicforms; this has been shown specifically for ciprofloxacin against S.aureus in addition to many other bacterial strains and antibioticclasses.¹⁴⁻¹⁷ Such studies have demonstrated that biofilms can be one toseveral orders of magnitude more resistant to the same antimicrobialagents as compared to their planktonic counterparts. This highlights theimportance of a bone-targeted approach for treating osteomyelitis, inorder to achieve higher local concentrations of antibiotic againstcausative biofilms and overcome potential resistance.

The specific bone-targeting properties of the BP family make these drugsideal carriers for targeting antibiotics to bone in osteomyelitispharmacotherapy.¹⁸⁻²⁰ BPs form strong bidentate or tridentate bonds withcalcium phosphate mineral, and as a result concentrate in hydroxyapatite(HA), particularly at skeletal sites of active metabolism includingsites of infection and inflammation.²¹ BPs also exhibit exceptionalstability against both chemical and biological degradation.²²BP-fluoroquinolone antimicrobial activity is complex and is related tothe specific strain of pathogen tested, the choice of antibiotic andcovalently bound BP moiety, the tether length between the twoconstituents, the bone binding affinity of the BP, theadsorption-desorption equilibria of the BP, and the stability/labilityand kinetics of the linkage moiety used for conjugation.¹⁸⁻²⁰ Therefore,accumulating evidence suggests that a ‘target and release’ linkerstrategy (FIG. 13) where a conjugate is stable in circulation, butlabile at the bone surface, may offer more opportunities foroptimization and success in this context. We thus hypothesized thatconjugation of ciprofloxacin to a phenyl BP moiety, throughmetabolically hydrolysable carbamate linkers, should mitigate theproblems seen with antibiotic dosing in osteomyelitis pharmacotherapy.The cleavable carbamate linkage is a key functionality in many drugsdesigned for target and release in specific tissues,^(23, 24) andconfers pharmacokinetic advantages such as stability in serum andlability at infected bone surfaces in the presence of an acidic andenzymatic environment (e.g. inflammation or infection).²⁵

A recent apparent success utilizing a bone-targeting and releasestrategy is provided by Morioka et al.²⁶ who designed an estradiolanalog conjugate using a cleavable variant (carbamate) of the morestable amide peptide bond. Several versions of this linkage wereattempted before the identification of a pharmacologically activevariant (aryl carbamate). Importantly, they demonstrated that a singledose of a similarly linked BP-estradiol conjugate (at a dose nearly5,600 times lower than the total dose of estradiol alone) produced asimilar effect on bone to that of the estradiol dosed alone.26 Theconjugate also provided an even greater therapeutic index, as there wereminimal effects systemically and in uterine tissues compared to theestradiol alone. Pharmacokinetic studies completed by Arns et al.²⁷ arein agreement with this dramatic enhancement of potency in studies basedon a BP-prostaglandin with a more labile linker. Other syntheticexamples of this approach in the antimicrobial field are reported forthe macrolide class;²⁸ however, only alkyl carbamates were explored andthe lack of further success suggests that target and release strategiesare likely chemical class-dependent (taking into considerationcompatibilities of the functional groups of each component) as well asbiochemical target dependent, and the design for any particular chemicalclass must be customized for its use.

In this Example the aryl carbamate BP-carbamate-ciprofloxacin conjugate6 (BV600022) is described and evaluated for its antimicrobial activityagainst common osteomyelitis pathogens and its in vivo safety andefficacy in an animal model of peri-prosthetic osteomyelitis. Thestudies in this Example utilize biofilm models and methodology, inaddition to planktonic cultures, to provide greater clinical ortranslational relevance.

At times herein, this compound may be referred to simply as “compound6,” “conjugate 6,” or simply “6.” Likewise, other compounds orconjugates may similarly be referenced as “compound e.g. 11”, “conjugatee.g. 11”, “or simply by the compound number designation (e.g. 11).

Results

Chemistry

An overall synthetic Scheme for 6 is shown in FIG. 16, starting from therelatively pharmacologically inert 4-hydroxyphenylethylidene BP (3). Thereagents for the Sheme presented in FIG. 16 were as follows: a Reagentsand conditions: (a) BTMS (2 equiv), Et2O, 0° C.—rt, 17 h, yield 95%. (b)i) tetraisopropyl methylene bisphosphonate (1 equiv), NaH (1 equiv),THF, rt, 10 min; ii) 1 (1 equiv), rt, 2 h, yield 52%. (c) Pd/C(Catalyst) (0.07 equiv), H2, MeOH, rt, overnight, yield 88%. (d)4-nitrophenyl chloroformate (1.1 equiv), Et3N (3 equiv), DCM, rt, 2.5 h,yield 44%. (e) Ciprofloxacin (1.2 equiv), water (pH 8.5), THF, 0° C.—rt,overnight, yield 52%. (f) i) BTMS (excess), DCM, 35° C., 24 h, ii) MeOH,rt, overnight, yield 86%.

The rationale for this BP design was to retain the bone-seeking abilityof the BP moiety while suppressing its unneeded antiresorptive activity,minimizing confounding factors to focus on evaluating the antimicrobialeffect due to the parent ciprofloxacin compound. BP ligands can also bedesigned to have antiresorptive functionality (of varying potency) ifneeded to provide a dual-action effect of bone tissue protection inaddition to antimicrobial effects at the anatomic site of infection.This phenyl BP was chosen with consideration of bone binding affinityand tether length, as previous studies have demonstrated that weakbinding affinity decreases targeting efficiency.^(13, 14) It waspostulated that the use of an aryl carbamate as a linker might offeroptimized stability in plasma and adequate release on bone for thisbiochemical target as compared to previously derived BP-fluoroquinoloneconjugates.

Additionally, a similar BP-ciprofloxacin conjugate having an amidelinkage as opposed to a carbamate linkage was synthesized as outlined inthe Scheme shown in FIG. 31 as a control conjugate 11 (BV600026). Thereagents for the Sheme presented in FIG. 31 were as follows: b Reagentsand conditions: (a) i) NaH (1.4 equiv), THF, 0° C.—rt, 1 h; ii) methyl4-(bromomethyl)benzoate (0.7 equiv), THF, 0° C.—rt, overnight, yield37%. (b) LiOH.H2O (5 equiv), MeOH, rt, overnight, yield 91%. (c) SOCl2(2 equiv), DMF (0.05 equiv), DCM, rt, 2 h, yield quantitative. (d)Ciprofloxacin (1 equiv), DIPEA (6 equiv), CHCl3, rt, overnight, yield65%. (e) i) BTMS (excess), DCM, 35° C., overnight, ii) MeOH, rt, 30 min,yield 82%. Previous investigations have indicated that amide conjugatesare not able to release the parent antibiotic and are thus lesseffective in vitro and in vivo, 11 which it was sought to verify in thisinstance.

Antibacterial Properties of BP-Ciprofloxacin Conjugates

Minimal Inhibitory Concentration (MIC) Assays:

The antimicrobial activity of both conjugates (6 and 11) and the parentantibiotic ciprofloxacin in standard laboratory planktonic culturesystems was evaluated against a panel of S. aureus clinical strainsassociated with bone infections, including methicillin-sensitive S.aureus (MSSA) and methicillin-resistant S. aureus (MRSA). FollowingEUCAST (European Committee on Antimicrobial Susceptibility Testing)guidelines^(,29) results from disc diffusion inhibition zone assaysrevealed diameters ranging from 25-40 mm (mean 31.5, SD±5), and everystrain demonstrated antimicrobial susceptibility to the parentantibiotic ciprofloxacin according to EUCAST clinical breakpoints.Minimal inhibitory concentration (MIC) results for 6 and 11 againsteight S. aureus strains using microdilution methodology are shown inFIG. 32. MICs for the parent compound ciprofloxacin were determinedconcurrently for reference (see FIG. 32) and were found to be consistentwith established clinical breakpoints.²⁹

Hydroxyapatite (HA) Binding Assay:

Having established the antimicrobial efficacy of 6, it was next soughtto evaluate HA binding ability. HA spherules were added to themicrobiological media and then introduced 6 at various concentrationssimilar to those used in the antimicrobial testing. Quantitativespectroscopic analysis of supernatant (without HA spherules) confirmedsignificant adsorption and retention of the conjugate by HA (FIGS. 18and 5).

pH Effect in Antimicrobial Susceptibility Testing (AST) on Planktonic S.aureus Strain ATCC-6538:

S. aureus strain ATCC-6538 was selected for further investigationbecause it demonstrated the lowest MIC profile for both ciprofloxacinand 6 (see FIG. 32) compared to the other strains tested. This ATCCstrain is also a well-known and robust biofilm-forming pathogen.Consequently, the conjugates were tested against a challenging pathogento limit bias and overestimated results, while also facilitatingassessment of antimicrobial activity in biofilm based and clinicallyrelevant models. Antimicrobial susceptibility testing (AST) onplanktonic S. aureus strain ATCC-6538 with 6 under both acidic andphysiological pH was performed to assess the effect of pH on conjugateactivity. Quantitative results from standard microdilution methodologyindicated that under acidic conditions (pH 5) the antimicrobial activityof 6 was improved overall as the MIC50 was reached at half the conjugateconcentration required to reach MIC50 under physiological conditions(FIGS. 6 and 4). These results and results presented demonstratedelsewhere herein, the minimum inhibitory concentration terms MIC50 orMIC90 refer to a reduction of 50% or 90% of bacterial load,respectively; and the biofilm-related terms of minimum biofilminhibitory concentrations (MBIC50 or MBIC90) refer to similar reductions(50% or 90%) but in biofilm bacterial load.

Time-Kill Assays of Compound 6:

Next, kinetic assays were performed with 6 according to CLSI (ClinicalLaboratory Standards Institute) methods.³⁰ Results indicated that thisconjugate was bactericidal at the previously established MIC formethicillin-susceptible (ATCC-6538) and methicillinresistant (MR4-CIPS)isolates of planktonic S. aureus within 1 hr and up to 24 hrs,preventing 100% of bacterial growth; these kinetic studies also revealedthat at half the MIC value, prevention of bacterial growth becameevident after 2 hrs and inhibition was at 50% of control after 24 hrs(e.g. FIG. 7).

Evaluation of Antimicrobial Efficacy of 6 Against Biofilms:

Compound 6 was then tested against pre-formed bacterial biofilms on twodifferent substrates (polystyrene and HA discs) to evaluateantimicrobial efficacy against biofilms, and to also determine ifsubstrate binding-specificity plays any role in the observedantimicrobial efficacy. Biofilms of S. aureus (ATCC-6538), andadditionally biofilms of P. aeruginosa (ATCC-15442), were grown onpolystyrene or HA as substrates and were subjected to varyingconcentrations of 6 for assessment of antimicrobial activity. P.aeruginosa here because it is a Gram negative pathogen and the secondmost common clinical pathogen in osteomyelitis, though less frequent inprevalence than Gram positive S. aureus. E.g. FIG. 8. shows results forpolystyrene as the substrate for biofilm growth, and the minimal biofilminhibitory concentration (MBIC50) of 6 was 15.6-31.2 μg/mL for S. aureusATCC-6538, which was comparable to the MIC for this strain in planktoniccultures. No MBIC50 was observed for P. aeruginosa ATCC-15442 in thetested range of concentrations and no MBIC90 was observed for eitherpathogen.

However, when HA discs were used as the biofilm substrate, markedbactericidal activity was observed with 6. As shown in FIG. 33, alltested concentrations of this conjugate resulted in statisticallysignificant (p<0.05, Kruskal-Wallis test) bactericidal activity andreduction of colony forming units (CFUs). The MBIC50 of 6 was 16 μg/mLand the MBIC90 was 100 μg/mL against S. aureus strain ATCC-6538; theMBIC90 for the parent drug ciprofloxacin was 8 μg/mL against thispathogen. However, against P. aeruginosa strain ATCC-15442 ciprofloxacinhad no inhibitory or bactericidal activity in this setting while theconjugate was bactericidal in acidic and physiological conditions at 50μg/mL, and showed improved bactericidal activity in physiologicalconditions as compared to S. aureus where improved antimicrobialactivity was observed in acidic conditions.

Preventative Antimicrobial Assays:

Next, antimicrobial tests with 6 were performed in a preventative typeof experimental setting with planktonic and biofilm cultures, whichcould also have clinical relevance in antibiotic prophylactic scenariosfor osteomyelitis pharmacotherapy. Here HA spherules were introduced tovarying concentrations of 6 and then inoculated with S. aureus for 24hrs, and quantitative assessments indicated no bacterial growth atconcentrations as low as 15.6 μg/mL and up to 250 μg/mL of 6, andminimal bacterial growth with strong inhibition at conjugateconcentrations ranging from 0.24 to 7.8 μg/mL as shown in e.g. FIG. 10.

Next, the amide conjugate (11) was tested for ability to treat S. aureusstrain ATCC-6538 biofilms in experimental conditions similar to thoseused to test the carbamate conjugate 6. When evaluating the activity of11 against established S. aureus biofilms grown on HA, and HA pretreatedwith 11 prior to biofilm growth in a preventative experimental setting,antimicrobial activity of 11 even at higher doses than those used totest 6, was insignificant in both cases as shown in FIG. 34.

When 6 was tested for ability to prevent S. aureus ATCC-6538 biofilmsfrom forming on pretreated HA, the conjugate showed superiorantimicrobial activity as compared the parent antibiotic and in contrastto 11 which showed no significant antimicrobial activity. FIG. 11 showsresults of quantitative biofilm cultures and CFU counts after 24 hrs ofgrowth, and at 100 μg/mL the parent drug ciprofloxacin inhibited allbiofilm growth whereas at 10 μg/mL, 6 inhibited all growth. Since themolecular mass of ciprofloxacin is approximately half that of 6, 6 was20 times more active in achieving complete bactericidal action ascompared to ciprofloxacin alone.

In Vivo Safety and Efficacy:

Since 6 demonstrated promising activity in vitro, we sought to assessdrug safety and efficacy in vivo in an animal model of periprostheticosteomyelitis. This model is a unique in-house jawbone peri-implantosteomyelitis model that was developed specifically for translationalvalue to study biofilm-mediated disease and host response in vivo.³¹Because a systemic treatment regimen is utilized, this assay also servesto model any infected bone surface, since the resulting osteolysisinvolved is key to attracting (targeting) high concentrations of aBPconjugate, like any high turnover site on bone, and to subsequentlyrelease the active ciprofloxacin component of the conjugate at thisdiseased bone surface. Briefly, biofilms of the jawbone osteomyelitispathogen Aggregatibacter actinomycetemcomitans (Aa; wild-type roughstrain D7S-1; serotype a), which is not indigenous to rat normal floraand specific to jawbone infections, were pre-inoculated on miniaturetitanium implants at 10⁹ CFU. To confirm Aa sensitivity to the parentdrug ciprofloxacin prior to our animal studies, AST and MIC assays wereperformed as performed for the long bone osteomyelitis pathogensdescribed previously. Disc diffusion inhibition zone assays revealeddiameters >40 mm, and the MIC90 was 2 μg/mL, indicating strongsusceptibility of this microbe to the parent drug ciprofloxacin. Aa hasalso been tested previously for susceptibility to a pH-sensitivebiotinylated ciprofloxacin prodrug and was found to be sensitive to theparent antibiotic.³² As with previous pathogens in this study, Aabiofilm pathogens grown on HA were tested for sensitivity to 6 and foundour conjugate displayed effective antimicrobial activity as shown inFIG. 35.

After Aa biofilms are established on implants in vitro, they aresurgically transferred to the jawbone of each rat. Animals areanesthetized, the cheeks are retracted and a transmucosal osteotomy isperformed so implants can be manually inserted into the osteotomy andsecured. Two biofilm-inoculated implants are placed in each rat (n=12rats, 24 implants total) in the palatal bone bilaterally. This modelallows standardized and reproducible quantities of viable bacteria to beformed as well-established biofilms on each implant, which we havepreviously demonstrated persists in vivo for several weeks afterplacement and causes infection, inflammation, and bone destructionlocally.³¹

Once peri-implant infection was established 1 week post-operatively, theanimals were dosed with 6, ciprofloxacin alone as a positive control,and sterile endotoxin-free saline as a negative control at the dosingregimens specified in the experimental section. To determine appropriatedosing concentrations, approximate initial doses were calculated for theconjugate based on previous studies and pharmacokinetic data usingsimilar target and release strategies also in rodents.^(13, 26)Increasing doses of 0.1, 1 and 10 mg/kg molar equivalents of 6 can allowfor determination of antimicrobial activity in 2 test animals per groupbased on sample size estimations and previous experience with the animalmodel.³² Animals were dosed via intraperitoneal injection under generalanesthesia, and all compounds were constituted in sterile physiologicalinjectable saline at appropriate pH. Intraperitoneal injection was usedbecause of the ease of administration in small rodents as compared withother parenteral methods like tail vein injection, and because thepharmacokinetics of ciprofloxacin following gastrointestinaladministration shows excellent bioavailability; serum drug levelsachieved after such administration are slightly less but comparable tothose with intravenous dosing with no substantial loss after first passmetabolism.³³ One week after pharmacotherapy, all animals weresacrificed and en bloc resection of peri-implant hard and soft tissueswas performed and homogenized for quantitative assessment of microbialload.

All animals tolerated the pharmacotherapy well with no cutaneousinjection-site reactions or inflammation. There were no signs of grosstolerability issues during therapy. Treatment efficacy wasquantitatively measured in terms of the logarithmic reduction of theamount of viable bacteria (mean log 10 CFU/gram of tissue) as shown inFIG. 36.

In vivo, the single dose of 6 at 10 mg/kg showed the highest efficacywith a 2 log reduction in bacterial count (99% bacterial killing) andnearly an order of magnitude greater activity than ciprofloxacin alonegiven at the same per dose concentration (mg/kg) but in multiple doses(30 mg/kg total dose). Thus, given the greater molecular weight of 6(˜2× of ciprofloxacin), the administered single dose of 6 at 10 mg/kgcould deliver roughly 5 mg/kg of effective ciprofloxacin assuming fullrelease, which is ⅙th of the ciprofloxacin molar dose of the controlciprofloxacin arm (30 mg/kg total). Ciprofloxacin alone in a multipledosing regimen resulted in a 1 log reduction in bacterial counts (90%bacterial killing). Concentrations of 6 at 0.1 and 1 mg/kg had littleeffect, suggesting that a minimum dose is necessary for clinical effectand that further chemistry optimization may be possible in this context.

To validate the animal study findings, and to provide for greater powerand larger sample size for statistical analysis, we conducted a secondanimal experiment nearly identical to the first except for allocation ofdosing regimens. Based on dosing data and antimicrobial results from ourfirst animal study described above, we focused this second animal studyon three treatment groups: negative control (n=5 rats), 6 at a singlehigh dose of 10 mg/kg (n=5 rats), and 6 at a multiple low dose regimenof 0.3 mg/kg 3×/week (n=2 rats). Dosing groups of 0.1 and 1 mg/kg wereexcluded as they showed no efficacy previously, and the parentantibiotic alone was also excluded since robust historical data existsfor ciprofloxacin efficacy which we also confirmed in our initial animalstudy. The multiple dosing regimen was utilized again to ascertainwhether the lack of recoverable bacteria could be attributed totreatment effect or experimental and sampling error. All otherexperimental parameters were identical to the first animal experiment,and each animal had two implants placed as before allowing for tworesults per animal and providing sufficient power for statisticalanalyses as determined by sample size estimations.

All animals again tolerated treatment and pharmacotherapy well and therewere no signs of gross tolerability issues during therapy. Clinicallyduring euthanasia and surgical resection, it was observed that themajority of the animals in the control group demonstrated evidence oflocalized peri-prosthetic inflammation as compared to the majority ofthe animals in the treatment groups which had non-inflamed peri-implanttissues, and implant retention was 23/24 implants (96%) which is a highretention rate and provided robust power for subsequent analyses.Quantitative antimicrobial results from this second animal experimentare shown in FIG. 37. Single factor ANOVA testing (a=0.05) comparingCFUs between treatment groups resulted in a p-value=0.006 forsignificance between groups, and post-hoc testing utilizing an unpairedt-test (p=0.0005; df=20) and Dunnett's multiple comparisons test(p<0.05) revealed significance for the single high dose of 6 treatmentas compared to the control, but not for the multiple low dose group(p>0.05) when compared to the control or to the single high dosetreatment group.

Discussion

Targeting antibiotics to bone by conjugation to a BP moiety (via areleasable carbamate linker) is a promising approach for the treatmentof osteomyelitis biofilms. Results of AST testing and MIC data presentedherein indicate that against planktonic S. aureus, ciprofloxacin and 6have effective bactericidal activity, and that the conjugation linkageimpacts antimicrobial activity of the parent drug as evidenced by theweaker activity of 11 (FIG. 32). Higher concentrations of 6 wererequired to reach MIC, which is anticipated since conjugation is achemical modification that can alter the biochemical interactions of theantibiotic prior to release from the conjugate. As a result, propertiesof the parent drug, including its pharmacodynamic effect, can be alteredby such modification. MIC results for 6 were consistent with previousliterature indicating that conjugates in this class can retain theantibacterial activity of the parent compound, although at slightlylower levels.^(9, 10)

Of interest was the wide distribution of MIC values for both conjugatesagainst tested S. aureus strains, as compared to ciprofloxacin alonewhich demonstrated little variance in antimicrobial efficacy against thesame strains (FIG. 17). There are several possible explanations forthese results. Different strains of bacteria within the same species areknown to show significant variance in terms of virulence andantimicrobial susceptibility/resistance to an antibiotic. It is wellestablished that strain-specific variances exist in antibiotic transportand efflux mechanisms, bacterial cell wall density, enzymatic activitylevels, resistance mechanisms, and ability to alter pH of theenvironment.³⁴ Ciprofloxacin bactericidal activity results fromintracellular inhibition of enzymes required for DNAreplication—topoisomerase II and IV.³⁵

It has been established that intact conjugates in this class generallylack significant intrinsic antibacterial activity,^(18, 19) and that anyBP-related antimicrobial effect is negligible; therefore, at leastpartial release of the parent drug is a prerequisite for significantantimicrobial activity, as observed with 6. This is consistent with thelow antimicrobial activity of 11 differing in its more stable amidelinkage, which resulted in 2-64× the concentration of the more labilecarbamate linked conjugate 6 to achieve the same antibacterial effect inthe assay.

After evaluating the antimicrobial efficacy of 6, it was sought toassess the bone-binding functionality of the BP moiety and foundeffective adsorption and retention to HA spherules by the conjugate in aconcentration-dependent manner. These results are consistent withpreviously reported analogs in this class containing BP moieties withsimilar bone affinities.^(13, 19) It was then tested whether activity of6 would vary in different pH conditions and found a slightly improvedprofile in acidic conditions, which may be explained at least partiallyby the fact that the linker is more labile at pH 5 than at pH 7.4 thusreleasing more ciprofloxacin at the lower pH. This could be useful forclinical osteomyelitis applications where biofilm pathogens along withhost inflammation and osteoclastogenesis produce an acidic local milieu.Other investigators have suggested, however, that although acidic pHbrought on by infecting organisms and inflammation could result in somedrug release in bone, the efficacy of such a process in providing asignificant concentration of the antimicrobial agent is doubtful, andthat prodrug design, conjugation scheme, and susceptibility to localenzymatic hydrolysis likely have greater impact on linker cleavabilityand efficacy.¹³ The data in this Example also support such conclusions.

Investigation of time-kill kinetics for 6 demonstrated an efficient rateof bactericidal activity against tested bacteria with sustainedbactericidal activity over 24 hrs, supporting cleavage activity of theparent antibiotic with a steadily sustained release profile over time.The antibiotic release kinetics observed here may be different thanthose observed with currently used biodegradable and non-biodegradabledelivery systems for osteomyelitis therapy, which generally demonstratean initial high bolus of antibiotic release at the site with a smallerpercentage of the remaining antibiotic dissipating over an extendedperiod of time.^(36, 37)

This Example presents evidence for antimicrobial efficacy of conjugatessuch as 6 in biofilm-relevant models in vitro and in vivo forosteomyelitis treatment. When osteomyelitis biofilms (S. aureus and P.aeruginosa) were grown in vitro on different substrates such aspolystyrene or HA, and then treated with 6, the conjugate was moreeffective against biofilms in the presence of HA versus polystyrene.This indicates that substrate binding-specificity plays a role inantimicrobial activity in addition to factors like strain of pathogentested and mode of bacterial growth (planktonic versus biofilm). Thefact that 6 was effective against osteomyelitis pathogens on HA, but noteffective against the same strains on polystyrene as a substrate,indicates that to effectively treat osteomyelitis biofilms, it isnecessary to bind to the substrate (e.g. HA) and release antibioticdirectly underneath or within a biofilm rather than just flow theantibiotic along the biofilm surface (as was the case with the parentantibiotic alone or 6 on polystyrene where no substrate binding occursand no activity was seen against established surface biofilms). Theimproved activity of 6 found in experimental settings using HA discs incomparison to the setting using polystyrene as a substrate is likely dueto the fact that the BP moiety of the conjugate possess high affinity toHA structures, and therefore bacteria adhering to HA were likelysubjected to a relatively higher concentration of the parent antibioticdue to localization of 6 to the disc. Also, cleavage of 6 at bone underbiofilm bacterial cells may be similar to carbamate cleavage underosteoclast cells as previously shown,22 suggesting that the localenvironment plays a role in this context and further indicating that theenvironment under bacteria, that also causes osteolysis, hassimilarities to the environment under osteoclasts on bone since theseenvironments both seem to be able to cleave the aryl carbamate linkageto release the active ciprofloxacin, probably due to a combination of pHand enzymatic hydrolysis. Previous work by Arns et al.²⁷ with BP(radiolabeled) prostaglandin conjugates suggest that, as with mostBPs,³⁸ the half-life of the conjugate in the bloodstream is less than 15minutes. Thus, in that time the conjugate is either bound to bone orexcreted. This research study also demonstrates that the half-life ofrelease of the active drug (prostaglandin in this case) from the BP onthe bone surface, with linkages related to our carbamate is between 5and 28 days. The linkage demonstrated herein must release closer to the5-day half-life to achieve the exciting in vivo result reported here.Arns et al and others27 have speculated that the mechanism of cleavageis most likely enzymatic under bone cells. In the presence of bacteriaon mineral surfaces, it is also likely to be an enzymatic-basedcleavage. As is already noted in the manuscript during in vitroantimicrobial studies devoid of osteoclasts, our carbamate basedconjugate is active, but our non-cleavable amide-based conjugate is farless active.

The conjugates were also tested in osteomyelitis preventativeexperiments against S. aureus, and found that 6 was 20 times more activein achieving complete bactericidal action as compared to ciprofloxacinalone (FIG. 11), whereas any antimicrobial activity of 11 was notdetectable (FIG. 34). These findings support an efficient mechanism ofcleavage and release over time of the parent antibiotic from 6 ascompared to 11. Efficient binding to HA and release of the parentantibiotic is requisite for conjugates in this class to demonstratesubstantial antimicrobial efficacy.

Finally, it was sought to test in vivo safety and efficacy of 6 in ajawbone peri-implant osteomyelitis rat model using the model jawbonepathogen Aa. To confirm Aa sensitivity to the parent drug ciprofloxacinprior to our animal studies, we performed in vitro AST and MIC assays asperformed for the long bone osteomyelitis pathogens in this study. Aademonstrated strong susceptibility to the parent drug ciprofloxacin. Aabiofilms grown on HA (similar to S. aureus and P. aeruginosa) were alsotested for sensitivity to 6 and found our conjugate displayed effectiveantimicrobial activity (FIG. 35). Therefore, two consecutive animalexperiments were performed utilizing a peri-implant jawboneosteomyelitis model. In the first in vivo study, a single dose of 6 at10 mg/kg showed the highest efficacy with 2 log reduction of CFU or 99%bacterial killing and nearly an order of magnitude greater activity thanciprofloxacin alone given at the same per dose concentration (mg/kg) butin multiple doses (FIG. 36), comparable or better than the parentantibiotic alone,¹⁸⁻²⁰ as was observed with the more labile 6 but notwith the more stable 11 even at high doses of exposure, confirming thatcleavage contributes and in some instances can be necessary forantimicrobial efficacy. Lower concentrations of 6 in this experimentwere ineffective. To validate these results we performed a second largerand more statistically powered in vivo experiment focusing on theefficacious dosing regimen (10 mg/kg) of 6 as compared to control andmultiple dosing regimens of 6. Again greatest CFU reduction and efficacywas observed at the single high dose (10 mg/kg) of conjugate.

In vivo experiments confirmed the ability of 6 at a safe and adequatesingle dose to target infected peri-implant bone and generate asufficient concentration of the parent antibiotic for bactericidalactivity against established Aa biofilms when the activity of the parentantibiotic alone had already diminished. As microbial quantificationinvolved an en bloc resected tissue homogenate, even biofilm bacteriawithin canaliculi of the 3-dimensional osseous architecture are includedfor analysis and not just surface pathogens (as the methodology did notinvolve surface scraping for plating and assessment). This suggestsefficacious BP absorption/adsorption to peri-prosthetic bone andantibiotic release as evidence by the considerable reductions in CFU ofbiofilm bacteria.

These results along with other studies in this field are also indicatingthat direct comparisons between these conjugates and their parentcompound are somewhat arbitrary as conjugates have unique pharmacometricparameters and predominantly localize to bone due to the BP moiety. Thisis in contrast to the parent antibiotics (the fluoroquinolone class ingeneral) which demonstrate much greater muscle and tendon uptake thanbone uptake in humans,³⁹ and thus correlate with adverse events such asAchilles tendon rupture in susceptible populations. Any futurepharmacokinetic modeling and testing for conjugates in this class shouldinclude a skeletal compartment of distribution mathematically, which isnot generally done with ciprofloxacin and most other antibioticpharmacokinetic studies. The importance of such an approach in humanpopulations for accurately determining bone pharmacokinetics of BP drugshas been established.⁴⁰ Such approaches will provide more accurate andnecessary pharmacological data in this context and also inform clinicaldosing approaches.

Materials and Methods

All manipulations were performed under nitrogen atmosphere unless statedotherwise. Anhydrous ethyl ether, anhydrous tetrahydrofuran, anhydrouscitric acid, chloroform, and magnesium sulfate were purchased from EMD.4-Benzyloxy benzyl alcohol, bromotrimethylsilane, 4-nitrophenylchloroformate, hydrochloric acid (37%), anhydrous ethanol, anhydrousN,N-dimethylformamide, and thionyl chloride were purchased from SigmaAldrich. Sodium sulfate was purchased from Amresco. Sodium hydride(57-63% oil dispersion), tetraisopropyl methylenediphosphonate, 10%Palladium on activated carbon, 4-(bromomethyl)benzoate, lithiumhydroxide monohydrate, and N-ethyldiisopropylamine were purchased fromAlfa Aesar. Ethyl acetate, hexane, and dichloromethane were purchasedfrom VWR. Anhydrous methyl alcohol, trimethylamine, and sodium carbonatewere purchased from Macron. Hydrogen gas was purchased from Airgas.Ciprofloxacin was purchased from Enzo Life Sciences. Acetonitrile (HPLCGrade) was purchased from Spectrum. All reagents were used as received,unless stated otherwise. All solvents were dried using 3 Å molecularsieves (20% m/v).⁴¹ Silica gel was purchased from Silicycle (SilicaFlashP60, 40-63 Å, 40-63 μm, 230-400 mesh).

Nuclear magnetic resonance spectra were recorded on Varian 400-MR2-Channel NMR Spectrometer with 96-spinner sampler changer and analyzedusing TopSpin and MestReNova. Chemical shifts (δ, ppm) for 1H werereferenced to residual solvent peaks. Mass spectra were obtained on aThermo-Finnigan LCQ Deca XP Max mass spectrometer equipped with an ESIsource under positive and/or negative modes using Tune Plus version 2.0software for data acquisition and Xcalibur® 2.0.7 for data processingand reported in m/z. Organic Elemental Analysis was performed on Flash2000 Elemental Analyzer by Thermo Fisher Scientific.

The purities of the final compounds 6 and 11 as well as commercialciprofloxacin were ≥95% and were determined using 1H, 31P NMRspectrometry, HPLC and Elemental Analyzer. Analytical HPLC of finalcompounds were performed on a SHIMADZU HPLC system equipped with diodearray detector. LabSolution software was used for both data collectionand analysis. HPLC Method A: Phenomenex Luna 5μ C18(2) 100 Å analyticalcolumn (250×4.6 mm) operating at a flow rate of 1.0 mL/min was used. Thefollowing solvent gradient was employed: (Buffer A=20% ACN in 0.1 MNH4OAc (pH 7.53), Buffer B=70% CAN in 0.1 M NH4OAc (pH 7.16)) 0-7 min 0%B, 7-25 min 100% B, 25-100 min 100% B.

Synthesis. 1-(Benzyloxy)-4-(bromomethyl)benzene (1)

4-Benzyloxy benzyl alcohol (1.00 g, 4.67 mmol) was dissolved inanhydrous diethyl ether (25 mL) in an oven-dried flask under nitrogen.The flask was cooled in an ice bath. Bromotrimethylsilane (BTMS) (1.26mL, 9.52 mmol, 2 equiv) was added by syringe. The flask was allowed toslowly warm to room temperature. After 17 hrs of stirring, the reactionmixture was poured into water (50 mL) and the organic phase wasseparated. The aqueous phase was washed with diethyl ether (2×20 mL) andthen the combined organic phase was washed with brine (2×20 mL) anddried over sodium sulfate. Evaporation of the solvent afforded compound1 as a white crystalline solid (1.23 g, 95% yield). 1H NMR (400 MHz,Chloroform-d) δ 7.47-7.28 (m, 7H), 6.98-6.90 (m, 2H), 5.07 (s, 2H), 4.50(s, 2H).

Tetraisopropyl (2-(4-(benzyloxy)phenyl)ethane-1,1-diyl)bis(phosphonate)(2)

Under nitrogen protection, anhydrous THF (2 mL) was added to sodiumhydride (57-63% dispersion in mineral oil) (75 mg, 1.80 mmol, 1 equiv).Tetraisopropyl methylene diphosphonate (570 μL, 1.80 mmol, 1 equiv) wasadded dropwise with stirring at room temperature. Gas was evolved andthe grey suspended solid was consumed leaving a clear solution. Themixture was stirred a further 10 min. Compound 1 (500 mg, 1.80 mmol, 1equiv) was added in one portion under nitrogen counterflow. The solutionremained clear for 1 min and then became turbid. Stirring was maintainedfor 2 hrs and the reaction progress was monitored by TLC (100% EtOAcvisualized by UV and cerium ammonium molybdate (CAM) stain). Thereaction mixture was poured into 5% aqueous citric acid (30 mL) andextracted with ether (2×30 mL), washed with brine (30 mL) andevaporated. The residue was purified by flash chromatography using aEtOAc:Hexane gradient (10-100%) to afford 2 as a colorless oil (0.508 g,52% yield). 1H NMR (400 MHz, Chloroform-d) δ 7.44-7.27 (m, 5H), 7.18 (d,J=8.6 Hz, 2H), 6.87 (d, J=8.7 Hz, 2H), 5.04 (s, 2H), 4.86-4.63 (m, 4H),3.15 (td J=16.6, 6.1 Hz, 2H), 2.44 (tt, J=24.2, 6.1 Hz, 1H), 1.48-1.01(m, 24H). 31P NMR (162 MHz, Chloroform-d) δ 21.11.

Tetraisopropyl (2-(4-hydroxyphenyl)ethane-1,1-diyl)bis(phosphonate) (3)

Compound 2 (0.508 g, 0.925 mmol) was dissolved in 13 mL of methanol and10% palladium on activated carbon (70 mg, 0.066 mmol, 0.07 equiv) wasadded. The flask was flushed with nitrogen then hydrogen, and stirredovernight with a hydrogen balloon in place. The reaction mixture wasfiltered through celite with 100 mL of methanol. Evaporation of thefiltrate gave the desired compound 3 as a slightly yellow oil (0.368 g,88% yield) that was used without further purification. 1H NMR (400 MHz,Chloroform-d) δ 7.07 (d, J=8.2 Hz, 2H), 6.69 (d, J=8.2 Hz, 2H), 4.71 (m,4H), 3.11 (td, J=16.9, 6.0 Hz, 2H), 2.47 (tt, J=24.4, 6.0 Hz, 1H),1.32-1.21 (m, 24H). 31P NMR (162 MHz, Chloroform-d) δ 21.06.

4-(2,2-Bis(diisopropoxyphosphoryl)ethyl)phenyl (4-nitrophenyl) carbonate(4)

Compound 3 (7.91 g, 15.9 mmol) was dissolved in 150 mL ofdichloromethane then triethylamine (6.70 mL, 47.9 mmol, 3 equiv) wasadded followed by p-nitrophenyl chloroformate (3.54 g, 17.6 mmol, 1.1equiv) in one portion. Reaction mixture was stirred for 2.5 hrs whilebeing monitored with TLC (5% MeOH in EtOAc, UV visualization). Afterdisappearance of starting material reaction was stopped and the targetcompound was purified by flash chromatography (1:1 ethyl acetate:hexane)to afford compound 4 (4.33 g, 44% yield). 1H NMR (400 MHz, Chloroform-d)δ 8.29 (d, J=9.1 Hz, 2H), 7.46 (d, J=9.1 Hz, 2H), 7.33 (d, J=8.5 Hz,2H), 7.15 (d, J=8.6 Hz, 2H), 4.84-4.58 (m, 4H), 3.22 (td, J=16.5, 6.2Hz, 2H), 2.47 (tt, J=24.1, 6.2 Hz, 1H), 1.33-1.14 (m, 24H).

7-(4-((4-(2,2-Bis(diisopropoxyphosphoryl)ethyl)phenoxy)carbonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (5)

Ciprofloxacin (2.76 g, 8.34 mmol, 1.2 equiv) was suspended in 74.7 mL ofwater in a flask. Then 8.30 mL of 1 M HCl was added and the flask wasstirred to dissolve ciprofloxacin, giving a clear colorless solution.Na₂CO₃ was added to adjust the pH to 8.5 and a thick white precipitateformed. The flask was placed in an ice bath and Compound 4 (4.28 g, 6.95mmol, 1 equiv) dissolved in 83 mL of THF was added slowly over about 5min. The flask was then removed from the ice bath, protected from lightand stirred overnight at room temperature. The reaction mixture wasconcentrated under vacuum to approximately half the original volume andfiltered through a fine glass frit funnel. The retained solid was washedwith water until no yellow color remained. The solids were thendissolved and washed from the frit with DCM, and the solution was loadedonto a flash silica column and eluted with MeOH:DCM gradient (2-5%) toafford compound 5 (3.47 g, 51.5% yield) as a white solid. 1H NMR (400MHz, Methanol-d4) δ 8.79 (s, 1H), 7.93 (d, J=13.3 Hz, 1H), 7.54 (s, 1H),7.30 (d, J=8.4 Hz, 2H), 7.05 (d, J=8.5 Hz, 2H), 4.70 (dpd, J=7.4, 6.2,1.3 Hz, 4H), 3.90 (m, 4H), 3.65 (s, br, 1H), 3.39 (s, br, 4H), 3.18 (td,J=16.6, 6.4 Hz, 2H), 2.65 (tt, J=24.3, 6.3 Hz, 1H), 1.43-1.34 (m, 2H),1.34-1.19 (m, 24H), 1.18-1.10 (m, 2H). 31P NMR (162 MHz, Methanol-d4) δ20.71. MS (ESI+) m/z: 808.2 (M+H), 830.2 (M+Na) calc. forC38H53FN3O11P2+: 808.3.

1-Cyclopropyl-7-(4-((4-(2,2-diphosphonoethyl)phenoxy)carbonyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (6).^(42, 43)

Compound 5 (10.0 mg, 1.24 μmol) was dissolved in DCM (200 μL) in a 1.5ml glass vial and BTMS (200 μL, 1.52 mmol, 122 equiv) was added and thevial was quickly capped and immersed in a 35° C. oil bath. Afterstirring for 24 hrs, solvent and BTMS were removed under vacuum and 1 mLof MeOH was added and the vial stirred overnight. Solvent was removedunder vacuum to afford pure compound 6 as a pale yellow solid with greenfluorescence (6.82 mg, 86.1% yield). 1H NMR (400 MHz, Deuterium Oxide) δ8.51 (s, 1H), 7.92 (d, J=12.2 Hz, 1H), 7.67 (s, 1H), 7.47 (d, J=8.3 Hz,2H), 7.10 (d, J=8.3 Hz, 2H), 3.98 (s, 2H), 3.79 (s, 2H), 3.67 (s, 1H),3.42 (s, 4H), 3.16 (td, J=15.5, 6.8 Hz, 2H), 2.21 (tt, J=6.9, 21.6 Hz,1H), 1.37 (d, J=6.9 Hz, 2H), 1.15 (s, 2H). 31P NMR (162 MHz, DeuteriumOxide) δ 19.16 MS (ESI−) m/z: 638.06 (M−H) calc. for C26H27FN3O11P2−:638.1. HPLC (Method A, UV 190, 274, 330 nm): tr=11.62 min.

Methyl 4-(2,2-bis(diisopropoxyphosphoryl)ethyl)benzoate (7).⁴⁴

Under nitrogen atmosphere, in a 25 mL round bottom flask, THF (5 mL) wasadded to 57-63% dispersion of sodium hydride in mineral oil (0.163 g,4.07 mmol, 1.4 equiv). The suspension was cooled to 0° C., whilestirring, and tetraisopropyl methylenediphosphonate (0.926 mL, 2.90mmol, 1 equiv) was added gradually. The reaction was allowed to reachambient temperature and once hydrogen gas stopped bubbling out of thereaction mixture, the solution was cooled to 0° C. again. Methyl4-(bromomethyl)benzoate (0.465 g, 2.03 mmol, 0.7 equiv) was dissolved inTHF (2 mL) and added to the reaction dropwise. The resulting solutionwas stirred overnight while slowly reaching ambient temperature. Thereaction mixture was then cooled to 0° C. and quenched with EtOH (1 mL).A 5% aqueous solution of citric acid in water (30 mL) was added and themixture was extracted with Et2O (3×30 mL), combined organics were washedwith brine (50 mL), dried on Na₂SO₄, filtered, concentrated underreduced pressure, and purified by silica gel column chromatography usinga EtOAc:Hex gradient (10-100%) to afford 7 as a faint yellow oil (0.371g, 37.0% yield). 1H NMR (400 MHz, Chloroform-d) δ 7.93 (d, J=8.0 Hz,2H), 7.33 (d, J=8.4, 2H), 4.79-4.68 (m, 4H), 3.88 (s, 3H), 3.24 (td,J=16.0, 6.4 Hz, 2H), 2.50 (tt, J=24.0, 6.2 Hz, 1H), 1.34-1.24 (m, 24H).31P NMR (162 MHz, Chloroform-d) δ 20.57.

4-(2,2-Bis(diisopropoxyphosphoryl)ethyl)benzoic Acid (8).⁴⁴

To a solution of 7 (0.131 g, 0.278 mmol) in MeOH (1.5 mL) in a 8 Dramglass vial, LiOH.H2O (0.058 g, 1.39 mmol, 5 equiv) was added and theresulting solution was stirred at room temperature overnight. Thereaction mixture was evaporated to dryness, the residue was dissolved inwater (30 mL), and HCl(aq) (1 M) was added slowly to reach pH 3. Theresulting mixture was extracted with CHCl₃ (3×30 mL). Combined organicswere dried on MgSO4 and concentrated under reduced pressure to afford 8as a thick clear oil (0.115 g, 90.6% yield). 1H NMR (400 MHz,Chloroform-d): δ=7.96 (d, J=8.0, 2H), 7.37 (d, J=8.0, 2H), 4.82-4.74 (m,4H), 3.28 (td, J=16.6, 6.1, 2H), 2.60 (tt, J=24.2, 6.2, 1H), 1.33-1.26(m, 24H). 31P NMR (162 MHz, Chloroform-d) δ 20.57.

Tetraisopropyl(2-(4-(chlorocarbonyl)phenyl)ethane-1,1-diyl)bis(phosphonate) (9)

Under nitrogen atmosphere, Compound 8 (0.162 g, 0.339 mmol) wasdissolved in chloroform (1 mL) an a catalytic amount of DMF (1.30 μL,0.017 mmol, 0.05 equiv) was added. Thionyl chloride (49.2 μL, 0.678mmol, 2 equiv) was added slowly and the reaction was allowed to stir for2 hrs at room temperature. Solvents were removed under vacuum to afford9 as clear oil. The product was immediately used in the next stepwithout further manipulation (quantitative yield).

7-(4-(4-(2,2-Bis(diisopropoxyphosphoryl)ethyl)benzoyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate(10)

Ciprofloxacin (0.112 g, 0.339 mmol, 1 equiv) was suspended in chloroform(1 mL) and N,N-diisopropylethylamine (DIPEA) (354 μL, 2.03 mmol, 6equiv) was added. Freshly made compound 9 (168 mg, 0.338 mmol, 1 equiv)was dissolved in chloroform (1 mL) and gradually added to theciprofloxacin:DIPEA suspension. The reaction mixture was covered withfoil and stirred at room temperature overnight. The following day,solvents were removed under vacuum and the resulting crude was dissolvedin DCM (5 mL) and filtered through a medium grade frit funnel and washedwith more DCM (3×5 mL). The filtrate was concentrated under vacuum andfurther purified by silica gel column chromatography using a MeOH:DCMgradient (0-10%) to afford 10 as a viscous oil that gradually solidified(0.226 g, 65.1% yield, 1.8 eq DIPEA salt). 1H NMR (400 MHz,Chloroform-d) δ=8.79 (s, 1H), 8.06 (d, J=12.8, 1H), 7.38 (m, 5H),4.80-4.73 (m, 4H), 4.00 (s, br, 4H), 3.56-3.53 (m, 1H), 3.33-3.20 (m,6H) 2.50 (m, 1H), 1.45-1.38 (m, 2H), 1.32-1.25 (m, 24H), 1.23-1.19 (m,2H). 31P NMR (162 MHz, Chloroform-d) δ 20.77.

1-Cyclopropyl-7-(4-(4-(2,2-diphosphonoethyl)benzoyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (11).42, 43

In a 8 Dram glass vial, compound 10 (0.108 g, 0.136 mmol) was dissolvedin DCM (700 μL) and BTMS (686 μL, 5.20 mmol, 38 equiv) was added. Thevial was capped and heated overnight at 35° C. while covered with foiland stirring. The following day, solvent was removed under vacuum andthe crude was quenched with MeOH (2 mL). The resulting solution wasstirred at room temperature for 30 min. Solvent was removed under vacuumto afford an orange oil. A few drops of water were added to produce ayellow solid. More MeOH (2 mL) was added and the resulting suspensionwas filtered using a medium grade fritted glass funnel. The resultingsolid was further washed with MeOH to afford 11 as a yellow powder(0.070 g, 82.0% yield). 1H NMR (400 MHz, Deuterium Oxide, pH 7.5):δ=8.54 (s, br, 1H), 7.90-7.87 (m, 1H), 7.65-7.63 (m, 1H), 7.54 (d,J=8.0, 2H), 7.44 (d, J=8.0, 2H), 4.79 (m, overlap with D20, 4H), 4.00(s, br, 2H), 3.79 (s, br, 2H), 3.47 (s, br, 3H), 3.34 (s, br, 2H), 3.21(td, J=14.0, 6.4, 2H), 2.30 (tt, J=22.0, 6.6, 1H), 1.38-1.33 (m, 2H),1.15 (s, br, 2H). 31P NMR (162 MHz, Deuterium Oxide, pH 7.5) δ 19.12. MS(ESI−) m/z: 622.24 (M−H) calc. for C26H27FN3O10P2−: 622.12. HPLC (MethodA, UV 190, 274, 330 nm): tr=4.43 min.

Antibacterial Properties of Bisphosphonate-Ciprofloxacin Conjugates

Experimental Strains:

Seven S. aureus clinical osteomyelitis strains ofmethicillin-susceptible profile and one of methicillin-resistant profilewere tested. These pathogens are part of the strain collection of theDepartment of Pharmaceutical Microbiology and Parasitology WroclawMedical University, Poland. Additionally, the following American TypeCulture Collection (ATCC) strains were chosen for experimental purposes:S. aureus 6538 and P. aeruginosa 15442.

Ha Discs:

For custom disc manufacturing, commercially available HA powder wasused. Powder pellets of 9.6 mm in diameter were pressed without abinder. Sintering was performed at 900° C. The tablets were compressedusing the Universal Testing System for static tensile, compression, andbending tests (Instron model 3384; Instron, Norwood, Mass.). The qualityof the manufactured HA discs was checked by means of confocal microscopyand microcomputed tomography (micro-CT) using an LEXT OLS4000 microscope(Olympus, Center Valley, Pa.) and Metrotom 1500 microtomograph (CarlZeiss, Oberkochen, Germany), respectively.

Disc Diffusion Test to Evaluate Sensitivity of Tested Strains toCiprofloxacin:

This procedure was performed according to EUCAST guidelines.29 Briefly,0.5 McFarland (MF) of bacterial dilution was spread on Mueller-Hinton(MH) agar plate. The discs containing 5 mg of ciprofloxacin wereintroduced and the plate was subjected to incubation at 37° C./24 hrs.Next, inhibition zones were recorded using a ruler. Obtained values (mm)were compared to appropriate values of inhibition zones from EUCASTtables.²⁹

Evaluation of the MIC of Tested Compounds Against Planktonic Forms ofClinical Staphylococcal Strains Analyzed:

To assess the impact of parent antibiotic and conjugates on microbialgrowth, 100 μl of microbial solutions of density 1×105 CFU/ml wereplaced into wells of 96-well test plates together with appropriateconcentrations of tested compounds. Immediately after that, theabsorbance of solutions was measured using a spectrometer (ThermoScientific Multiscan GO) at 580 nm wavelength. Subsequently, plates wereincubated for 24 hrs/37° C. in a shaker to obtain optimal conditions formicrobial growth and to prevent bacteria from forming biofilms. Afterincubation, the absorbance was measured once again. The followingcontrol samples were established: negative control sample one: sterilemedium without microbes; negative control sample two: sterile mediumwithout microbes implemented with DMSO (dimethyl sulfoxide,Sigma-Aldrich) to final concentration of 1% (v/v); positive controlsample one: medium+microbes with no compound tested; positive controlsample two: medium+microbes with no compound tested but implemented withDMSO to final concentration of 1% (v/v). Rationale for use of 1% DMSOwas that ciprofloxacin dissolves efficiently in this solvent, however,concentrations of DMSO>1% could be detrimental for microbial cells. Toassess relative number of cells, the following calculations wereperformed. The value of absorbance of control samples (medium+microbesfor conjugate, medium+microbes+DMSO for ciprofloxacin) was estimated at100%. Next, the relative number of cells subjected to incubation withtested compounds were counted as follows: value of control sampleabsorbance/value of tested sample*100%.

To confirm results obtained by spectrophotometric assessments, treatedbacterial solutions were transferred to 10 mL of fresh medium and leftfor 48 hrs at 37° C. The occurrence of opacification or lack ofopacification of media was proof of pathogen growth or lack of growth,respectively. Additionally, bacterial solutions were cultured on theappropriate stable medium. Growth or lack of growth of bacterialcolonies together with above-mentioned results from liquid culturesserved as confirmation of results obtained spectrophotometrically.

Spectroscopic Analysis of 6 and 11 in Tryptic Soy Broth (TSB)Microbiological Media with the Addition of HA Spherules:

Various conjugate concentrations were introduced to HA powder(spherules) suspended in TSB microbiological medium. Solutionscontaining BP-ciprofloxacin an HA spherules were introduced to wells ofa 24-well plate. Final concentration of powder was 10 mg/1 mL, whilefinal concentration of conjugates was 0.24-250 mg/L. Immediatelyafterward the absorbance of solutions was measured using a spectrometer(Thermo Scientific Multisca GO) at 275 nm wavelength. Plates were shakenautomatically in the spectrometer prior to assessment. Next, plates wereleft for 24 hrs/37° C./shaking. After 24 hrs, absorbance was measuredonce again. To assess the relative concentration of the conjugate at 0hr and 24 hrs, values of absorbance taken in the beginning and at theend of experiment were compared. The excitation slit, emission slit,integration time, and increment were optimized based on theconcentration of samples.

Antimicrobial Susceptibility Testing of 6 Against Planktonic Cultures ofS. aureus Strain ATCC-6538 in Acidic Versus Physiological pH:

This experimental setting was performed in the same manner as describedpreviously for disc diffusion testing, but microbiological media wasadjusted to pH 7.4 and pH 5 using KOH or HCL solution and measured usinga universal pH-indicator (Merck, Poland).

Time-Kill Assay for 6 Against S. aureus Strain ATCC-6538 (MSSA) andClinical MRSA Strain (MR4-CIPS):

This experiment was performed in the same manner as described previouslyunder the subheading: “Evaluation of MIC of tested compounds againstplanktonic forms of clinical staphylococcal strains analyzed”, butabsorbance assays (at 580 nm wavelength) were taken in hour: 0, 1, 2, 4,8, 16, and 24.

Antimicrobial Susceptibility Testing of 6 Against Preformed Biofilms ofS. aureus Strain ATCC-6538 and P. aeruginosa Strain ATCC-15442:

Strains cultured on appropriate agar plates (Columbia agar plate for S.aureus; MacConkey agar plate for P. aeruginosa) were transferred toliquid microbiological media and incubated for 24 hrs/37° C. underaerobic conditions. After incubation, strains were diluted to thedensity of 1 MF. The microbial dilutions were introduced to wells of24-well plates containing HA discs as a substrate, or simply topolystyrene wells where the bottom surface of the wells served as thesubstrate for biofilm development. Strains were incubated at 37° C. for4 hrs. Next, the microbe-containing solutions were removed from thewells. The surfaces, HA discs and polystyrene plates, were gently rinsedto leave adhered cells and to remove planktonic or loosely-boundmicrobes. Surfaces prepared in this manner were immersed in fresh TSBmedium containing 0.24-125 mg/L of 6 and ciprofloxacin as a control.After 24 hrs of incubation at 37° C. the surfaces were rinsed usingphysiological saline solution and transferred to 1 mL of 0.5% saponin(Sigma-Aldrich, St Louis, Mo.). The surfaces were vortex-mixedvigorously for 1 minute to detach cells. Subsequently, all microbialsuspensions were diluted 10-1 to 10-9 times. Each dilution (100 mL) wascultured on the appropriate stable medium (MacConkey, Columbia for P.aeruginosa and S. aureus, respectively) and incubated at 37° C. for 24hrs. After this time, the microbial colonies were counted and the numberof cells forming biofilm was assessed. Results were presented as themean number of CFU per square millimeter surface ±standard error of themean. To calculate the surface area of HA discs, x-ray tomographicanalysis was applied. For estimation of the area of test plate bottoms,the equation for circle area: πr² was applied.

Preventative Ability of 6 and 11 to Inhibit S. aureus 6538 Adherence toHA:

Various concentrations of 6 and 11 were introduced to HA powder(spherules) suspended in TSB microbiological medium. Solutionscontaining 6 and HA spherules were introduced to wells of 24-wellplates. Final concentrations of powder were 10 mg/1 mL, while finalconcentrations of the conjugate were 0.12-250 mg/L. Suspensions wereleft for 24 hrs/37° C./shaking. After 24 hrs, suspensions were removedfrom the wells and impulse-centrifuged to precipitate HA powder. Next,supernatant was very gently discarded and a fresh 1 mL of S. aureus ofdensity 105 CFU/mL was introduced to the HA spherules. Subsequently,this solution was shaken, absorbance was measured using 580 nmwavelength and left for 24 hrs/37° C./shaking. After incubationabsorbance was measured again and values from 0 hr and 24 hrs werecompared to assess reduction of bacterial growth with regard to controlsample one (bacterial suspension but no spherules) and control sampletwo (bacterial suspension+spherules but with no conjugate added).Additionally, solutions were impulse centrifuged, the supernatant wasgently discarded, while bacteria-containing HA spherules were cultureplated as before and quantitatively assessed. For testing of 11,solutions containing HA spherules and higher concentrations of 11ranging from 1-400 μg/mL and ciprofloxacin concentrations ranging from0.5-400 μg/mL were prepared and again compared to the control sample(bacterial suspension but no HA) for ability to inhibit biofilmformation. Higher concentrations of 11 were tested because of thedemonstrated weaker activity of an amide conjugate as compared to thecarbamate conjugate.

Survival of S. aureus after 24 Hrs of Incubation on HA Pretreated with6:

HA discs were immersed in 2 mL of solution containing variousconcentrations of BP-ciprofloxacin or ciprofloxacin alone and left for24 hrs/37° C. HA discs incubated in DMSO or phosphate buffer served ascontrol samples. Next, discs were rinsed 3 times with sterile water.After rinsing, 2 mL of 0.5 MF of. S. aureus ATCC6538 were introduced towells containing HA discs as a substrate for biofilm development andbiofilms were formed as before.

Ethics Statement:

All animal protocols and procedures were approved and performed inaccordance with the Institutional Animal Care and Use Committee (IACUC)of the University of Southern California (USC), and in accordance withthe Panel on Euthanasia of the American Veterinary Medical Association.USC is registered with the United States Department of Agriculture(USDA), has a fully approved Letter of Assurance (#A3518-01) on filewith the National Institutes of Health (NIH) and is accredited by theAmerican Association for the Accreditation of Laboratory Animal Care(AAALAC). The title of our IACUC approved protocol is: “Bone targetedantimicrobials for biofilm-mediated osteolytic infection treatment”, andthe protocol number is 20474. All animal protocols, and investigatorsand staff involved in the animal studies presented herein, adhered tothe Guide for the Care and Use of Laboratory Animals, the USDA AnimalWelfare Regulations (CFR 1985) and Public Health Service Policy onHumane Care and Use of Laboratory Animals (1996).

In Vivo Animal Study:

For this study 12 five-month-old, virgin, female Sprague-Dawley ratsweighing approximately 200 g each were used in this study. Two to threeanimals were housed per cage in a vivarium at 22° C. under a 12-hrlight/12-hr dark cycle and fed ad libitum with a soft diet (PurinaLaboratory Rodent Chow). All animals were treated according to theguidelines and regulations for the use and care of animals at USC.Animals were under the supervision of fulltime veterinarians on call 24hrs/day who evaluate the animals personally on a daily basis. All animalexperiments are described using the ARRIVE45 guidelines for reporting onanimal research to ensure the quality, reliability, validity andreproducibility of results.

This animal model is an in-house jawbone peri-implant osteomyelitismodel designed specifically to study biofilm-mediated disease and hostresponse in vivo.³¹ Biofilms of the jawbone osteomyelitis pathogen Aawere pre-formed on miniature titanium implants at 10⁹ CFU. To confirm Aasensitivity to the parent drug ciprofloxacin prior to our animalstudies, AST and MIC assays were performed against planktonic Aa inaddition to the biofilm HA assay as described for the long boneosteomyelitis pathogens. After biofilms were established on the implantsin vitro, they were surgically transferred to the jawbone of each rat.For surgery, animals were anesthetized with 4% isoflurane inhalantinitially followed by intraperitoneal injection of ketamine (80-90mg/kg) plus xylazine (5-10 mg/kg). Then local anesthesia was given viainfiltration injection of bupivicaine 0.25% at the surgical site.Buprenorphine sustained release (1.0-1.2 mg/kg) was then givensubcutaneously as preemptive analgesia before making initial incisions.Once anesthetized, the buccal mucosa of each rat was retracted and atransmucosal osteotomy was performed with a pilot drill into thealveolar ridge in the natural diastema of the anterior palate. Implantswere then manually inserted into the osteotomy and secured into the boneuntil the platform is at mucosal level. Two biofilm-inoculated implantswere placed in each rat (n=12 rats) in the palatal bone bilaterally.

One week post-operatively isoflurane 4% was given again to brieflyanesthetize the rats and check implant stability and document clinicalfindings at the implant and infection site, such as presence or absenceof inflammation. The animals were then dosed via intraperitonealinjection with BP-ciprofloxacin (6 at 0.1 mg/kg, 1 mg/kg, or 10 mg/kg asa single dose, and at 0.3 mg/kg 3×/week for a multiple dosing group) orciprofloxacin alone (10 mg/kg 3×/week also as a multiple dosing group)as a positive control, and sterile endotoxin-free saline as a negativecontrol.

Allocation of animals to treatment and control groups was done through arandomization process. The multiple dosing group animals wereanesthetized as before prior to each additional injection over thecourse of the week. All compounds were of pharmacological grade andconstituted in sterile physiological injectable saline at appropriatepH. One week after pharmacotherapy, all animals were euthanized in a CO₂chamber (60-70% concentration) for 5 minutes, followed by cervicaldislocation. Resection of peri-implant tissues (1 cm2) was performed enbloc and implants were removed. Clinical parameters were noted atsurgery and resection, such as presence or absence of peri-prostheticinflammation. Rat allocations to treatment and control groups weredeidentified and concealed from subsequent investigators analyzing themicrobial data.

For microbial analysis, resected peri-implant soft tissue and bone washomogenized and processed immediately after surgical resection byplacement in 1 mL of 0.5% saponine and vortexed for 1 min before beingserially diluted. Serial dilutions at a dilution factor of 10 (e.g. 0.1mL of saponine solution transferred to 0.9 mL of 0.9% sterile isotonicsaline solution) ranging from 10⁰ to 10⁻⁹ were prepared, and 0.1 mL ofsolution from each of the dilutions was cultured on plates using aspread plate method. The medium for culturing Aa consisted of modifiedTSB, and frozen stocks were maintained at −80° C. in 20% glycerol, 80%modified TSB. All culturing was performed at 37° C. in 5% CO2 for 48hrs. The numbers of viable Aa bacteria cultured (number of CFUs per gramof tissue) was counted manually and the reduction in the mean log₁₀number of CFU per gram as a function of treatment was recorded. In orderto confirm Aa bacterial morphotype and also rule out contamination, Gramstaining and histologic evaluation was performed by sampling of coloniesfrom plates once CFU counting was completed.

Statistical Analysis

Statistical calculations were performed with SPSS 22.0 (IBM, Armonk,N.Y.) and Excel 2016 (Microsoft Corporation, Redmond, Wash.). Poweranalyses were performed to determine sample size estimation for in vitroand in vivo studies prior to experimentation using G Power 3 software.⁴⁶Quantitative data from experimental results for each group was analyzedfirst with descriptive statistics to understand the distribution of thedata (parametric or non-parametric) and to generate the mean, standarderror, standard deviation, kurtosis and skewness, and 95% confidencelevels. The data was then analyzed using the Kruskall-Wallis test orone-way ANOVA as applicable and statistical significance was accepted atp<0.05 when comparing treatments to controls. Additionally, for in vivoexperiments, post-hoc testing using unpaired t-tests and Dunnett's testfor multiple comparisons was performed.

Abbreviations Used

Aa, Aggregatibacter Actinomycetemcomitans; AAALAC, American Associationfor the Accreditation of Laboratory Animal Care; ANOVA, Analysis ofvariance; ARRIVE, Animal Research: Reporting of In Vivo Experiments;AST, antibiotic sensitivity test; ATCC, American Type CultureCollection; BP, bisphosphonate; BTMS, bromotrimethylsilane; CFU, colonyforming units; CLSI, Clinical Laboratory Standards Institute; EUCAST,European Committee on Antimicrobial Susceptibility Testing; HA,hydroxyapatite; IACUC, Institutional Animal Care and Use Committee; MBC,mean bactericidal concentrations; MBIC50, minimal biofilm inhibitoryconcentration required to inhibit the growth of 50% of organisms; MF,McFarland; MH, Mueller Hinton; MIC50, minimal inhibitory concentrationrequired to inhibit the growth of 50% of organisms; MSSA,methicillin-sensitive S. aureus; Pd/C, palladium on activated carbon;SD, standard deviation; BTMS, bromotrimethylsilane; DCM,dichloromethane; SOCl₂, thionylchloride; SEM, scanning electronmicroscopy.

REFERENCES FOR EXAMPLE 5

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Example 6

Carbamate-linked bisphosphonate-ciprofloxacin is demonstrated herein tobe a viable antimicrobial conjugate for, inter alia, targeted therapy ofinfections bone disease (FIG. 14).

Bisphosphonates (BPs) can form strong bi- and tri-dentate interactionswith calcium and thus target bone or hydroxyapatite (HA) surfaces (wherebiofilm pathogens also reside). The feasibility of abone-biofilm-targeting antimicrobial approach was demonstrated bysuccessfully designing, synthesizing, and testing abisphosphonate-carbamate-ciprofloxacin (BCC, compound 6) conjugate invitro and in vivo against common bone biofilm pathogens. Our resultsindicated that BCC (compound 6) has a strong bactericidal profileagainst common long bone and jawbone osteomyelitis organisms in vitro,particularly when biofilm models were used with HA as the substrate formicrobial growth and antimicrobial testing. Biofilm growth on HA wasinhibited by chemisorbed BCC (compound 6) in an osteomyelitispreventative experimental setting, where the conjugate demonstrated apredictable rate of sustained release and was 20 times more active inachieving complete bactericidal action as compared to the parent drugciprofloxacin alone. Efficacy and safety of BCC (compound 6) againstbiofilms of Aggregatibacter actinomycetemcomitans was demonstrated invivo in an animal model of jawbone peri-implantitis. In vivo, a singleintraperitoneal dose of 10 mg/kg (15.6 μmol/Kg) of the conjugateproduced 99% peri-implant bactericidal efficacy, demonstrating an orderof magnitude greater activity than the parent antibiotic ciprofloxacinalone given in multiple doses (90.6 μmol/Kg, totaling a 6-fold higheroverall dose of ciprofloxacin). At this single dose of 10 mg/kg, BCC(compound 6) showed greater efficacy and disease resolution than thehigher multiply dosed parent antibiotic ciprofloxacin, with no potentialsystemic toxicity or adverse effects owing to the pharmacokinetic andpharmacodynamic advantages of bone targeting/biodistribution andsustained antibiotic release, respectively, at the site of biofilminfection.

Dental implants are a critical part of modern dental practice and it isestimated that up to 35 million Americans are missing all of their teethin one or both jaws. The overall market for these implants to replaceand reconstruct teeth is expected to reach $4.2 billion by 2022. Whilethe majority of implants are successful, some of these prosthetics faildue to peri-implantitis, leading to supporting bone destruction.Peri-implantitis has a bimodal incidence, including early stage (<12months) and late stage (>5 years) failures; both of these criticalfailure points are largely the result of bacterial biofilm infections onand around the implant. Peri-implantitis is a common reason for implantfailure. Dental implants failures are generally caused by biomechanicalor biological/microbiological reasons. The prevalence ofperi-implantitis, the most severe form of microbiological-relatedimplant disease leading to the destruction of supporting bone isdifficult to ascertain from the current literature. However, recentstudies indicate that peri-implantites is a growing problem withincreasing prevalence⁴. A recent study of 150 patients followed 5 to 10years showed a rate of peri-implantitis of approximately 17% and 30%respectively, indicating that it is a significant issue⁵. Early implantfailure or lack of osseointegration is a separate problem and occurs inroughly 9% of implanted jaws⁶. This is more prevalent in the maxilla⁶and is associated with bacterial infection during surgery or from anearby site (e.g. periodontitis) as well as other well-recognized andmodifiable risk factors such as smoking, diabetes, excess cement, andpoor oral hygiene².

Biofilm infection can be involved in the etiophathogeneiss ofperi-implantitis. Biofilm infections represent a unique problem fortreatment and are often difficult to diagnose, resistant to standardantibiotic therapy, resistant to host immune responses, and lead topersistent intractable infections⁷. The biofilm hypothesis of infectionhas been steadily expanded since the early elucidation that bacterialive in matrix supported communities^(8,9). It is now established thatover 65% of chronic infections are caused by bacteria living inbiofilms⁷. This implies that approximately 12 million people in the USare affected by, and almost half a million people die in the US eachyear, from these infections. Peri-implantitis and periodontitis areamong the most common biofilm infections encountered. Peri-implantitishas been found to be a comparatively simpler infection with less diversecommunities (and keystone pathogens) than periodontitis infections¹⁰.Typically, gram negative species predominate¹¹. Other orthopedic orosseous infections including those of the jaw, are also caused bybacterial biofilm communities¹² making the technology developed hereamenable for use in these diseases as well.

Currently treatment approaches to peri-implantits have theirlimitations. While peri-implantitis has several causes, the predominantetiology is bacterial biofilm. There are no universally acceptedguidelines or protocols for peri-implantitis therapy, many of theclinical regimens for bacterial peri-implantitis treatment compriselocal and systemic antibiotic delivery¹³ and surgical debridement of thelesion, including restorative grafting with bone graftsubstitutes^(14,15). Clinical experience has shown, however, that it isdifficult to advance even a local antibiotic delivery device to thebottom of a deep peri-implant pocket and to infected jawbone, or to getsystemic antibiotics to penetrate adequately into infected jawbone tokill biofilm pathogens¹⁶, which is largely due to the intrinsic poorbone (and peri-implant) biodistribution or pharmacokinetics of theantibiotics¹⁷. In previous long-term studies, even when infectedimplants were cleaned locally with an antiseptic agent and systemicantibiotics were administered, there was additional loss of supportingbone in more than 40% of the advanced peri-implantitis lesions¹⁵.

In addition, longer-term systemic antibiotic therapy could result insystemic toxicity or adverse effects, and also resistance. Therefore ithas become common practice by clinicians to use local delivery systemsfor achieving higher therapeutic antibacterial concentrations in bone.For example, dentists use chairside mixing of minocycline or doxycyclinepowder (e.g. Arestin®), or chlorhexidine solution (e.g. PerioChip®),with bone graft material for local delivery¹⁸. Such approaches aremerely a slurry and do not represent a strong binding between theantibiotic and the bone substitute as in the BioVinc approach, and thussuffer from comparatively earlier washout and less efficientpharmacokinetics as previously discussed. In addition, investigatorshave also used several biodegradable and non-biodegradable localantibiotic delivery systems¹⁹. However, these approaches have severallimitations, e.g., non-biodegradable approaches (e.g.polymethylmethacrylate cements) require a second surgery to remove theantibiotic loaded device, are incompatible with certain antibiotics, andsuffer from inefficient release kinetics; in some cases, <10% of thetotal delivered antibiotic is released¹⁷. Biodegradable materialsincluding fibers, gels, and beads are receiving increasing interest,however, their clinical efficacy for the treatment of peri-implantitisis not well-documented³. Even when effective antimicrobials/antisepticsare used to treat peri-implantitis in the jaw, such as localchlorhexidine delivery, there is minor influence on treatment outcomesas demonstrated in prospective animal and human studies^(15,17). Thesedata taken together further support the poor pharmacokinetics ofantibiotics in bone as previously mentioned, and highlight the need forbone-binding/bone-targeted and sustained antibiotic release strategies.

BP-Conjugates

Considering the limitations of current treatment approaches, it is asignificant advance in the field to develop a bone/biofilm-targetingantimicrobial agent. The BP-antibiotic (BP-Ab) conjugates providedherein can overcome many challenges associated with poor antibioticpharmacokinetics or bioavailability in bone and within bone-boundbiofilms. These compounds can reduce infection via a “targeting andrelease approach,” which can reduce concern with systemic toxicityand/or drug exposure in other (e.g. non-infected) tissues. The BP-Abconjugates can be integrated into a bone graft substitute. The BP-Ab canbe a BP-fluoroquinolone conjugate. In some instances, the BP-Ab can be abisphosphonate-carbamate-ciprofloxacin (BCC, compound 6), as shown inFIG. 15. The exemplary structure of FIG. 15 is also referred to hereinas BCC (compound 6). When integrated into a bone graft the BP-Ab bonegraft material can also be referred to as a BP-Ab-bone graft. Forexample, when the antibiotic is a fluoroquinolone, it can be referred toas a BP-FQ-bone graft. These compound(s) can effectively adsorb tohydroxyapatite (HA)/bone, and can achieve a sustained release andantimicrobial efficacy against biofilm pathogens over time. Thecompounds and graft material integrating the compound(s) provided hereincan be used as an anti-infective bone graft substitute for adjuncttreatment or prevention of peri-implantitis. The conjugate will bereleased locally from the graft material with sustained release kineticsand cleaved in the presence of bacterial or osteoclastic activity as wehave previously demonstrated, in vitro and in vivo, in other resultsprovided elsewhere herein. In this way the grafts can provide greaterlocal concentrations of the FQ, such as ciprofloxacin, as compared tocurrent delivery routes. In sum the compounds and bone-graft materialsprovided herein can contain an antibiotic that is conjugated to a safeor pharmacologically inactive (non-antiresorptive) BP moiety bound tocalcium/HA in the graft material via strong polydentate electrostaticinteractions, and the antibiotic releases over time; it does not simplyrepresent a topical antibiotic that is merely mixed in as a slurry withexisting bone graft material as some current clinical approaches in thiscontext. This chemisorbed drug attached to calcium phosphate mineral(HA) is therefore a major advance in the field and overcomes many of thelimitations in antibiotic delivery to peri-implant bone for effectivebactericidal activity against biofilm pathogens.

The general concept of targeting bone by linking active drug moleculesto BPs has been discussed in a review³⁰. However, as of this time no FDAapproved drugs have been developed, as early attempts led to eithersystemically unstable prodrugs or non-cleavable conjugates that werefound mostly to inactivate either component of the conjugate byinterfering with the pharmacophoric requirements. In the quinolone fielda prominent example was described by Herczegh where antibacterialproperties of the fluoroquinolone were diminished upon conjugation witha stable BP-linked congener³¹⁻³². Therefore, a target and release linkerstrategy is needed.

Recently, medicinal chemistry strategies exploiting less stable linkingtechniques start to emerge. Others have linked fluoroquinolones via thecarboxylic acid group to several different BP moieties. They found thatglycolamide ester prodrugs of the antibiotics moxifloxacin andgatifloxacin reduced infection when used prophylactically in a ratosteomyelitis model³³. This same group has used acyloxycarbamate andphenylpropanone based linkers to tether the same antibiotics via aminefunctionality to simple BP systems³⁴. They show using the sameprophylactic rat model that these conjugates are also better than theparent antibiotic at inhibiting the establishment of infection. TheTarganta team³³ has carried several of these prodrug strategies on intouse with the glycopeptide antibiotic oritavancin³⁵. This dual functiondrug seems to be somewhat effective in preventing infection. However, todate they have not published studies showing that they can treat anestablished infection and they also have not published pharmacokineticsof the prodrug. It is believed that these analogs are too labile in thebloodstream to fully realize success with this therapeutic approach astheir drug candidate selection was based in part on plasma instability.Thus it is believed that these compounds developed by these groups failto achieve effective local concentrations of the antibiotic.

The BCC compound(s) (FIG. 15) can incorporate the phenyl moiety of thephenyl carbamate linker directly into the BP portion of the molecule.Release kinetics can be modified or tuned via modification of the phenylring with electron withdrawing or donating groups, which can alter theliability of the linker. Additionally, the BP core lacks effectivenessas an antiresporptive agent, and thus, does not carry the risk ofmedication-related osteonecrosis of the jaw like the more potentnitrogen-containing BP drugs (e.g., zoledronate^(39,40). It isdemonstrate herein and in other Examples herein that this target andrelease strategy using the phenyl carbamate linker very likely releasesthe active drug directly into the bacterial biofilm in the bone milieu.The bone targeting is so effective that it works better thanciprofloxacin against biofilms grown on HA bone matrix surrogate than onplanktonic cultures grown in plastic vessels. An analog conjugate madewith a non-cleavable amide linkage (bisphosphonate-amide-ciprofloxacin,BAC, compound 11), leaving out the phenolic oxygen of the carbamate, wasfound to have very little effect on bacterial growth under anycircumstances, demonstrating that active cleavage of the conjugate isrequired for antimicrobial activity.

A synthesis scheme for BCC (compound 6) is demonstrated in FIG. 16. Thecompound was characterized by ¹H, ¹³C and ³¹P NMR as well as by massspectrometry. In order to help determine if antimicrobial activity isprimarily due to the released ciprofloxacin we decided to synthesize anamide linked conjugate that was designed not to release ciprofloxacinfrom the conjugate as well. The synthesis of this compound went smoothly(FIG. 31), and afforded the control compound BAC (compound 11) withreasonable yield.

A series of assays were performed to determine the minimum inhibitoryconcentrations (MIC) of BCC (compound 6) (also referred to as “BCC(6)”), BAC (compound 11) (also referred to as “BAC (11)”) and the parentdrug ciprofloxacin against a group of Staphylococcus aureus (SA)strains. These experiments were carried out using dilution assaysaccording to the European Committee on Antimicrobial SusceptibilityTesting guidelines (ref. 43). Testing of the three compounds (FIG. 17)demonstrates that the BCC (compound 6) conjugate retains significantbactericidal activity against these pathogens while the BAC (compound11) has lost most of the activity. These antibiotic susceptibilitytesting (AST) and MIC data indicate that against planktonic andclinically relevant SA pathogens, ciprofloxacin and BCC (compound 6)have strong bactericidal activity, and that the conjugation linkingimpacts antimicrobial activity of the parent drug as evidenced by theweak activity of the BAC (compound 11). Our testing of ciprofloxacinagainst these strains was consistent with established clinicalbreakpoints.

The compounds were tested for adsorption to suspended HA beads as asurrogate for bone binding since HA is the main inorganic constituent inbone. Our BCC (compound 6) is indeed taken up by the HA beads asindicated by the measurement of residual conjugate in the supernatantafter bead removal (FIG. 18). Conjugate in the supernatant was measuredat 0 and 24 hrs by spectrophotometry using the absorbance at 275 nM thedetermined λ_(max) for BCC (compound 6). This clearly indicates that BCC(6) is bound to this bone surrogate. We did not measure the binding ofthe BAC (compound 11) as the data for BCC (compound 6) was consistentwith binding of this type of BP which would drive the BAC (compound 11)binding as well (ref. 35, 44).

The next in vitro test was to combine the bone surrogate targetingactivity of the BCC (6) with the ready release of ciprofloxacin that isindicated by the MIC activity against SA strains. For this experiment HAdiscs were pretreated with solutions of the conjugates or ciprofloxacinat the designated concentrations followed by rinsing to remove thecompound solution. Biofilms of SA (strain ATCC-6538) were allowed togrow according to our published procedures. Quantitative counts ofcolony forming units (CFUs) were carried out after 24 hrs of growth.Discs pretreated with DMSO and PBS were used as controls. The resultsdemonstrated that BCC (6) inhibits all bacterial growth at 10 μg/mL(FIG. 11) whereas the pure ciprofloxacin was completely inhibitory at100 μg/mL. Because the molecular weight of the conjugate isapproximately half that of the pure ciprofloxacin, this indicated thatthe BCC (compound 6) is roughly 20 times more potent at completelyinhibiting the growth of bacterial biofilms than the parent drug. Thissupports the release of drug from the conjugate over time in the milieuof bone matrix. The amide conjugate BAC (11) did not inhibit the growthof biofilms on bone substitute even at very high concentrations (datanot shown) indicating that the release of ciprofloxacin was crucial tothis activity and consistent with earlier literature and the planktonicculture studies (FIG. 18)^(30, 34, 35, 44).

With the aforementioned results showing that our BCC (6) hasbactericidal activity, we were ready to test its activity against abiofilm infection in an animal model. Briefly, Aggregatibacteractinomycetemcomitans (Aa; wild-type rough strain D7S-1; serotype a),which is not indigenous to rat normal flora and specific to jawboneinfections, are pre-inoculated on miniature titanium implants at 10⁹CFU. To confirm Aa sensitivity to the parent drug ciprofloxacin prior toour animal studies, we performed AST and MIC assays with Aa, asperformed for the long bone osteomyelitis pathogens describedpreviously. Disc diffusion inhibition zone assays revealed diameters >40mm, and the MIC⁹⁰ was 2 mg/mL, indicating strong susceptibility of Aa tothe parent drug ciprofloxacin. Inoculated implants, bearing the Aabiofilms, were placed into 12 rats (2 implants per animal). This modelreliably forms well-characterized biofilm infections on surroundingjawbone, causing inflammation and associated peri-implantitis disease.(ref. 45) After allowing biofilms to develop the animals were randomizedinto three treatment groups (BCC (6) 10 mg/kg single dose in 5 animals,BCC (6) 0.3 mg/kg 3× weekly in 2 animals, and control treatment withsterile saline in 5 animals). A pilot experiment (2 animals/group)demonstrated that the BCC (6) single dose at 10 mg/kg was approximatelyas effective as ciprofloxacin given at 30 mg/kg total dose administeredin a 3×10 mg per week regimen (not shown). Therefore, we decided not toinclude the ciprofloxacin control in the larger experiment to reduceanimal usage. All animals tolerated treatment and pharmacotherapy wellwith no adverse events. Clinically, during euthanasia and surgicalresection, we observed that the majority of the animals in the controlgroup demonstrated evidence of localized peri-prosthetic inflammation ascompared to the majority of the animals in the treatment groups whichhad non-inflamed peri-implant tissues, and implant retention was 23/24implants (96%) overall providing for robust statistical analyses.

Quantitative determination of the CFU of Aa from resected peri-implanttissue (23/24 implants) post-euthanasia was carried out and results areshown in FIG. 19 where the single dose of 10 mg/kg BCC (6) demonstratedapproximately 6 log units of kill and even the low multiple dose showed2-3 (99% to 99.9%) log kill in this experiment. With this experiment thesingle dose of 10 mg/kg BCC (6) showed the greatest efficacy and washighly statistically significant (p=0.0005) as compared to the controlarm.

In both of these animal models the BCC (6) was delivered byintraperitoneal injection to assure exposure to the compound since thereis relative bioequivalence with intravenous or gastrointestinal routesof administration of fluoroquinolones. We believe these results in totaldemonstrate the feasibility of using releasable BP-antibiotic (BP-Ab)conjugate as a drug for the treatment of peri-implant disease andrelated osteomyelitis. Here we propose to build on these results toincorporate the BP-Ab conjugate into a dental bone graft substitutematerial for local oral delivery and release.

Example 7

Design and synthesis of additional BP-Ab conjugates (FIG. 20).Additional BP-Ab conjugates can be designed using, for example,ciprofloxacin and moxifloxacin conjugated to BPs (e.g.4-hydroxyphenylethylidene BP (BP 1, FIG. 20), its hydroxy-containinganalog (BP 2, FIG. 20, with higher bone affinity) and pamidronate (BP 3,FIG. 20), via carbamate based linkers (e.g. carbamate, S-thiocarbamate,and O-thiocarbamate). FIG. 21 shows an exemplary synthesis scheme forsynthesis of BP-Ab conjugates with an O-thiocarbamate linker. Conjugateswith S-thiocarbamate linkage (slightly more labile) can be obtained byisomerization of conjugates with O-thiocarbamate linkage via theNewman-Kwart rearrangement (ref. 47, 48). Preliminary chemistry hasalready been conducted to demonstrate the feasibility of the quicksynthesis of these targets. Adding bone affinity is therefore welldemonstrated using the α-OH containing BPs (49). Added bone affinitywill enhance concentrations of the conjugate at the bone surface andfacilitate higher local concentrations of drug short term and long term.For the synthesis of conjugates with α-OH containing BPs (BP 2 andpamidronate, FIG. 20), since the α-OH bisphosphonate ester is prone torearrangement to a phosphonophosphate, the α-OH can be protected withthe tert-butyldimethylsilyl (TBS) group (Scheme 2, FIG. 22) (50). Thenthe α-O-TBS BP 2 ester are activated by 4-nitrophenyl chloroformate andreacted with ciprofloxacin or moxifloxacin similarly as in FIG. 21. Forα-O-TBS BP 3 ester, a linker with phenol group (e.g., linker 1(resorcinol), linker 2 (hydroquinone), linker 3 (4-hydroxyphenylaceticacid), FIG. 20) are used to tether BP and antimicrobial agents, and thesynthesis route using linker 3 is illustrated as an example here (Scheme3, FIG. 23). All BP-Ab conjugates are characterized by 1H, 31P, 13C NMR,MS, HPLC, and elemental analysis to assure identity.

The mineral binding affinity of the BP-Ab conjugates can be determined.Briefly, Anorganic bovine bone large particle size (uniformly 1-2 mm)can be accurately weighed (1.4-1.6 mg) and suspended in a 4 mL clearvial containing the appropriate volume of assay buffer [0.05% (wt/vol)Tween20, 10 μM EDTA and 100 mM HEPES pH=7.4] for 3 hr. This bonematerial can then be incubated with increasing amounts of BP-Ab (0, 25,50, 100, 200 and 300 μM). Samples can be gently shaken for 3 h at 37° C.in the assay buffer. Subsequent to the equilibrium period, the vials canbe centrifuged at 10,000 rpm for 5 min to separate solids andsupernatant. The supernatant (0.3 mL) can be collected and theconcentration of the equilibrium solution are measured using a ShimadzuUV-VIS spectrometer (275 nm wavelength). Fluorescent emission can alsobe used to calculate binding parameters. Nonspecific binding can bemeasured with a similar procedure in the absence of HA as control. Theamount of parent drug/BP-Ab conjugates bound to HA is deduced from thedifference between the input amount and the amount recovered in thesupernatants after binding. Binding parameters (K_(d) and Bmax representthe equilibrium dissociation constant and maximum number of bindingsites, respectively) can be calculated using the PRISM program(Graphpad, USA) and measured in 5 independent experiments. Compoundswith an equilibrium dissociation constant (K_(d)) lower than 20 μM (˜2×K_(d) of parent BPs) can be preferred. A two-sample t-test can be usedto evaluate the binding parameters of the BP-Abs. The sample size (n=5)in each group can be used to detect the effect size 1.72 for thishypothesis at a power of 80% and a one-side Type I error of 0.05.

The linkage-stability of the BP-Ab conjugates can be determined.Briefly, the linker stability of each BP-Ab conjugate can be tested inPBS buffers with different pH (pH=1, 4, 7.4, 10) and human or canineserum. BP-Ab can be suspended in 400 μL of above-mentioned PBS or in 400μL of 50% (v/v in PBS) human or canine serum. The suspension/solutioncan be incubated for 24 h at 37° C. and centrifuged at 13000 rpm for 2min, and the supernatant can be recovered. Methanol (5× volume relativeto supernatant) can be added to each supernatant, and the mixture can bevortexed for 15 min to extract released fluoroquinolone. The mixture canbe then centrifuged at 10000 rpm for 15 min to pellet the insolublematerial. The supernatant containing the extracted fluoroquinolone canbe recovered and evaporated to dryness. The dried pellets can beresuspended in PBS, and the amount of released fluoroquinolone can bedetermined by UV-VIS measurements as described previously. Thepercentage of fluoroquinolone drug released can then be calculated basedon the input amount and the measured amount of released drug. Theidentity of released drug can be confirmed by LC-MS analysis and/or NMRif the concentrations are sufficient.

The in vitro inhibition of biofilm growth on HA discs can be determined.Briefly, for custom disc manufacturing, commercially available HA powdercan be used. Powder pellets of 9.6 mm in diameter can be pressed withouta binder. Sintering can be performed at 900° C. The tablets can becompressed using the Universal Testing System for static tensile,compression, and bending tests (Instron model 3384; Instron, Norwood,Mass.). The quality of the manufactured HA discs can be checked by meansof confocal microscopy and microcomputed tomography (micro-CT) using anLEXT OLS4000 microscope (Olympus, Center Valley, Pa.) and Metrotom 1500microtomograph (Carl Zeiss, Oberkochen, Germany), respectively. HA discscan then be introduced to the following concentrations [mg/mL] of eachBP-Ab conjugate and ciprofloxacin/moxifloxacin: 800, 400, 200, 100, 50,25, 10, 5, 1 and left for 24 h/37° C. After incubation, HA discs can beremoved and introduced to 1 mL of PBS and left for 5 min in gentlerocker shaker; 3 subsequent rinsings are performed this way. Afterrinsing, 1 mL of Aa suspension can be introduced to discs and left for24 h/37° C. Discs can then be rinsed to remove non-bound bacteria andsubjected to vortex shaking. The serial dilutions of suspension obtainedcan then be culture plated on modified TSB agar plates and colony growthis counted after 24 h.

The oseeointegration effect of the BP-FQ-bone grafts on critical sizecan be evaluated in supra-alveolar peri-implant defect model for bonegrafting. Briefly, in this split mouth design, mandibular PM2-PM4 arebilaterally extracted in 6 beagle dogs (3 males, 3 females) and areallowed to heal for 12 weeks. Crestal incision are made followed bymucoperiosteal flap reflection. Ostectomy are performed to create a 6 mmsupra-alveolar defect. Implant site osteotomy preparations are made ineach of the premolar regions by sequential cutting with internallyirrigated drills in graduated diameters under copious irrigation.Implants (Astra Tech Osseospeed Tx® 3×11 mm) are placed in the positionof PM2-PM4 on each side in such manner that the implants are positioned4 mm supracrestally in relation to the created defect and at the samedistance from the buccal cortical bone plate. Dogs are divided randomlyinto 3 different groups (2 dogs per group):

1. Anorganic bovine bone (1 g large particle size 1-2 mm) chemisorbedwith BP-fluoroquinolone are used on the right side and collagen plugs(negative control) are used on the left side.

2. Anorganic bovine bone (1 g large particle size 1-2 mm, positivecontrol) are used on the right side and collagen plugs (negativecontrol) are used on the left side.

3. Bio-Oss® (1 g large particle size 1-2 mm) chemisorbed withBP-fluoroquinolone are used on the right side and Bio-Oss® (1 g largeparticle size 1-2 mm, positive control) are used on the left side.

Chemistry and antimicrobial assay results from experiments describedabove can inform calculations of the ideal standardized quantity of theconjugate for adsorption to graft material for use in all in vivoexperiments described here. Early calculations predicated based on thepreliminary results indicate that 5 mg or less of conjugate adsorbed to1 g of graft material will provide 2-3 orders of magnitude bactericidalactivity above the MIC of tested pathogens. Our BP-fluoroquinoloneconjugate can be applied in a range of bone graft materials includingcommercially available ones, e.g., Bio-Oss®; thus we choose house-madean organic bovine bone and BioOss as a positive control in the study fora demonstration of wide applications of the conjugate. All defects arefilled (depending on the groups above) with a standardized amount ofbiomaterial up to the platform of each implant on both sides, andBio-Gide® membranes are used to cover the graft and the implants forimproved stability. The flaps are closed in a tension free manner withthe use of periosteal releasing incisions, internal mattress and finallymarginal single interrupted sutures (PTFE 4,0, Cytoplast, USA). MicroCTare acquired at this point and animals are monitored clinically forinflammation and adverse events. Additionally, as described in theexperiments to follow, these animals undergo PK studies to assess forany systemic exposure to the components within the graft material (e.g.intact conjugate, BP, antibiotic, or linker). Animals are sacrificedafter 12 weeks and the mandibles are resected and examined by micro-CTfollowed by histologic preparation. Baseline micro-CT scans of the jawsare taken for comparison to post-experimental scans. Quantitative 3Dvolumetric micro-CT and histomorphometric analyses are performed toexamine the volume of new bone present in peri-implant sites, as well asfirst bone-to-implant contact, total defect area, regenerated area,regenerated area within total defect area, regenerated bone, residualbone substitute material, percentage of mineralized tissue, soft tissue,and void. Finally, necropsy are performed for post-mortem evaluation oforgans and systems for gross and microscopic signs of tolerabilityissues from local oral therapy.

Antimicrobial efficacy of the BP-FQ-bone grafts can be evaluated in acanine peri-implantitis model. Briefly, in this split mouth design,mandibular PM2-PM4 are extracted bilaterally in 8 beagle dogs (4 males,4 females; 48 teeth total) using minimally traumatic technique. After 3months of healing mucoperiosteal flaps are elevated on both sides of thejaw and osteotomy preparations are made in each of the premolar regionsby sequential cutting with internally irrigated drills in graduateddiameters under copious external irrigation. Using a non-submergedtechnique, implants (Astra Tech Osseospeed Tx® 3×11 mm) are installed ateach site. The sequence of implant placement are identical in both sidesbut randomized with a computer generated randomization scheme betweendogs. Healing abutments are connected to the implants and flapsapproximated with resorbable sutures. A plaque control regimencomprising brushing with dentifrice is then initiated four times a week.Twelve weeks after implant placement just prior to initiation ofexperimental peri-implantitis, microbiological samples are obtained fromall peri-implant sites with sterile paper points (Dentsply, Maillefer,size 35, Ballaigues, Switzerland) and placed immediately in Eppendorftubes (Starlab, Ahrensburg, Germany) for microbiological analysis.Microbiologic analysis are performed as we have previously detailed viaDNA extraction and 16S rRNA PCR amplification. (55) PCR amplicons aresequenced using the Roche 454 GS FLX platform and data analyzed with theQuantitative Insights into Microbial Ecology (QIIME) software package(56). Colony forming unit counts (CFU/mL) are determined from samples asin our Phase I study as described earlier. At this point experimentalperi-implantitis are initiated as follows. Aggregatibacteractinomycetemcomitans (Aa) biofilm, a keystone periodontal pathogen,which is not endogenous to canine flora, are initiated on the healingabutments in vitro as performed in our previous experiment in a ratanimal model and also in our previous animal peri-implantitis study. Thebiofilm inoculated healing abutments are placed on the implants andcotton ligatures are placed in a submarginal position around the neck ofimplants. After 10 weeks of bacterial infection, microbial sampling andanalysis are done again as before and micro-CT scans are taken as thebaseline for the peri-implantitis defect. Treatment of this experimentalperi-implantitis model are initiated by surgical debridement of allimplant sites by raising full-thickness buccolingual flaps, removing anyexisting calculus from implant surfaces using an air-powder abrasiondevice, and wiping of the implant surfaces with gauze soaked inchlorhexidine gluconate 0.12%. The animals are divided into 4 groups asfollows (2 dogs per group):

1. Anorganic bovine bone (1 g large particle size 1-2 mm) withchemisorbed BP-fluoroquinolone are used on the right side and collagenplugs (negative control) are used on the left side.

2. Anorganic bovine bone (1 g large particle size 1-2 mm, positivecontrol) are used on the right side and collagen plugs (negativecontrol) are used on the left side.

3. Anorganic bovine bone (1 g large particle size 1-2 mm) withchemisorbed BP-fluoroquinolone are used on the right side and anantimicrobial releasing device (100 mg topical minocycline, positivecontrol) are used on the left side.

4. Bio-Oss® (1 g large particle size 1-2 mm) with chemisorbedBP-fluoroquinolone (positive control) are used on the right side and anantimicrobial releasing device (100 mg topical minocycline, positivecontrol) are used on the left side.

Treatment group assignments are blinded to future investigators for dataanalysis. Standardized and comparable amounts of antimicrobials are usedin treatment groups. After treatment, flaps are repositioned and sutured(PTFE 4,0, Cytoplast, USA) and oral hygiene measures reinstituted after1 week following suture removal. Clinical and micro-CT scan examinationsare performed again at 3 months after surgery and also microbiologicalsamples are acquired at this time point for analysis as described above.Six months after peri-implantitis surgery animals are euthanized andmicro-CT scans are performed, and the jaws are resected for assessmentof histopathologic parameters as detailed in the section “critical sizesupra-alveolar peri-implant defect model.” An inflammatory score aredetermined from histologic sections as previously detailed (ref. 57) forcorrelation with clinical and radiologic findings.

Statistical analysis: Statistical calculations are performed with SPSS22.0 (IBM, Armonk, N.Y.) and Excel 2016 (Microsoft Corporation, Redmond,Wash.). Power analyses were performed to determine sample sizeestimations for all animal studies using G Power 3 software⁵⁸. Followingdata collection from these animal studies, quantitative outcomes areanalyzed first with descriptive statistics to understand thedistribution of the data (parametric or non-parametric) and to generatethe mean, standard error, standard deviation, kurtosis and skewness, and95% confidence levels. The data are analyzed using the Kruskall-Wallistest, ANOVA, or mixed linear models as applicable and statisticalsignificance are carried out at a=0.05 level when comparing groups.Post-hoc testing using unpaired t-tests and Dunnett's test for multiplecomparisons are also performed to further validate findings. All animalexperiments are described using the ARRIVE guidelines for reporting onanimal research to ensure the quality, reliability, validity, andreproducibility of results⁵⁹.

The drug compound and component stability and in vitro ADME of BCC (6)can be evaluated. This data can help establish if there is likely to beany large differences in human metabolism vs. experimental animals.Incubation of 6 with human, rat, and dog liver microsomes andhepatocytes followed by LC/MS analysis of the metabolite mixture areperformed. The metabolic profile of ciprofloxacin is known^(62,63), andso our focus are on any metabolites of the BP portion of the moleculeand of the parent (e.g. piperazine ring cleavage as is known forciprofloxacin). Once metabolites have been determined in vitro, plasmasamples from other in vivo experiments described above are used todetermine these compounds at steady state in vivo.

The toxicology of the BCC (6) can be evaluated in rat and dog todetermine NOAEL. In order to determine the NOAEL and maximum tolerateddosage (MTD) in rat and dog we first carry out dose ranging studies.Groups of 6 rats (3 males, 3 females), are given a single intravenousdose of 10 mg/kg for 6, or based on our best assessment at the time. Thedose are escalated by doubling until acute toxicity is noted (MTD) thenthis dose are reduced by 20% sequentially until no effects are seen,this will be the NOAEL for the compound. Toxicity are assessed as mild,moderate or substantial, and moderate toxicity in ≥2 or substantialtoxicity in ≥1 animal define the MTD⁶⁴. Animals are followed for bodyweight and clinical observations for 5 days. After 5 days, animals areeuthanized and necropsy performed to assess for organ weight andhistology (15 sections to include liver and kidney based on clinical BPtoxicology). A similar dose range study are carried out in dogs (1/sex,starting at the equivalent dose as determined from allometric scaling 4mg/kg assuming 250 g rats and 10 kg dogs) and include hematology andclinical chemistry in addition to identical terminal studies as in rat.This can use a total of 4-6 cohorts.

An expanded acute toxicity testing in groups of animals includingtoxicokinetics and recovery testing at the NOAEL and the MTD can beperformed. Groups of 48 rats including 10/sex can be used for each dosefor assessment of toxicity and 9/sex for toxicokinetics and 5/sex forrecovery. Toxicokinetics are determined at 6 time points (3 rats/timepoint chosen randomly from male or female) following administration ofeach dose. Time points are 5, 30, 60, 120 mins, 12 hrs, and 24 hrs postdosing. Recovery animals are observed for 14 days followed by assessmentof organ weight and histology as in the above study. From thetoxicokinetic study, PK parameters are determined by non-compartmentalanalysis (NCA) including Cmax, AUC and half-life. An identicalexperiment are carried out in canines but include 10 total animals(3/sex for dosing and 2/sex for recovery) with multiple blood draws fromeach animal at the same time points as for the rats. The AUC at theNOAEL for canines are used to calculate the maximum allowable exposurefrom the bone graft/BP-fluoroquinolone conjugate as described in aim 2and PK experiments in canines are used to determine if there is systemicexposure above 1/100 of this level.

For population modeling, a unique 3-compartment (blood/urine/bone)mathematical model of BP pharmacokinetics which has been validatedclinically and are applied to the current project⁶⁵. From the caninestudy, in each animal at the time of euthanasia, we sample bone (jaw andfemur), tendon (gastrocnemius) for determination of BP andfluoroquinolone concentrations. We combine these data and our model todescribe the time course in dogs. From this model we can simulate theexpected exposure of bone and cartilage to both BP and fluoroquinolonewith alternative dosing or repeated dosing. This can inform subsequenthuman dosing. The nonparametric adaptive grid (NPAG) algorithm withadaptive gamma implemented within the Pmetrics package for R (Laboratoryof Applied Pharmacokinetics and Bioinformatics, Los Angeles, Calif.) areused for all PK model-fitting procedures as previously described⁶⁶⁻⁶⁸.Assay error (SD) is accounted for using an error polynomial as afunction of the measured concentration, and comparative performanceevaluation are completed using Akaike's information criterion, aregression of observed versus predicted concentrations, visual plots ofPK parameter-covariate regressions, and the rule of parsimony.

Example 8

The BP-Ab conjugates can be integrated into grafts and grafting devices.In embodiments, one or more of the BP-Ab conjugates can be integratedinto an already approved bone graft product, such as the bovine bonematerials from BioOss® (Geistlich Pharma AG, Switzerland) or MinerOss®(BioHorizons, Birmingham, Ala.) to name a few. The BP-Ab conjugate(s)can be admixed with a support material for use as a dental bone graftsubstitute. The product will comprise the conjugate adsorbed to anorganic bovine bone material. This material will allow the localdelivery of antibiotic to the region of bone graft implantation toreduce bacterial infection rates and associated dental pathology such asperi-implantitis and other infections. The dental applications for ourproduct could include not only peri-implantitis treatment, but alsosocket preservation after tooth extraction, ridge or sinus augmentation,periodontitis prevention or treatment, osteomyelitis or osteonecrosistreatment or prevention, or other oral and periodontal surgeryapplications where such a bone graft could be beneficial. TheBP-fluoroquinolone conjugate material will be intimately adsorbed on thebone graft substitute and our preliminary data show sustained releaseinto the area of bone destruction in the case of infections, whichallows our product to more effectively deliver antibiotic to the site ofinfection with negligible to no systemic exposure to either component ofthe conjugate compound.

The grafting material can also be beneficial for non-dental graftingprocedures, such as sinus grafting procedures.

REFERENCES FOR EXAMPLES 6-8

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Example 9

This Example demonstrates various BP conjugate compounds and synthesisschemes. BP-carbamate-ciprofloxacin BP conjugate and synthesis scheme isdemonstrated in FIG. 16 and related descriptions.BP-carbamate-moxifloxacin BP conjugate and synthesis scheme isdemonstrated in FIG. 38. FIG. 39 shows a BP-carbamate-gatifloxacin BPconjugate and synthesis scheme. FIG. 40 shows a BP-p-HydroxyphenylAcetic Acid-ciprofloxacin BP conjugate and synthesis scheme. FIG. 41shows a BP-OH-ciprofloxacin BP conjugate and synthesis scheme. FIG. 42shows a BP-O-Thiocarbamate-ciprofloxacin BP conjugate and synthesisscheme. FIG. 43 shows a BP-S-Thiocarbamate-ciprofloxacin BP conjugateand synthesis scheme. FIG. 44 shows a BP-Resorcinol-ciprofloxacin BPconjugate and synthesis scheme. FIG. 45 shows aBP-Hydroquinone-ciprofloxacin BP conjugate and synthesis scheme.

FIG. 46 shows one embodiment of a genus structure for aBP-fluoroquinolone conjugate, where W can be O or S or N, X can be O, S,N, CH₂O, CH₂N, or CH₂S, Y can be H, CH₃, NO₂, F, Cl, Br, I, or CO₂H, Zcan be H, CH₃, OH, NH₂, SH, F, Cl, Br, or I, and n can be 1-5. FIG. 47shows various BP-fluoroquinolone conjugates.

FIG. 48 shows one embodiment of a genus structure for a genus of aphosphonate containing an aryl group, where X can be H, CH₃, OH, NH₂,SH, F, Cl, Br, or I, Y can be PO₃H₂, or CO₂H. Z can be OH, NH₂, SH, orN₃, and n can be 1 or 2. FIG. 49 shows various BPs, where X can be F,Cl, Br, or I and n can be 1 or 2.

FIG. 50 shows various BP's with terminal primary amines. FIG. 51 showsvarious BPs coupled to a linker containing a terminal hydroxyl and aminefunctional groups where R can be Risedronate, Zoledronate, Minodronate,Pamidronate, or Alendronate. FIG. 52 shows variousBP-pamidronate-ciprofloxacin conjugates. FIG. 53 shows variousBP-Alendronate-ciprofloxacin conjugates.

Example 10

The antimicrobial properties of a thiocarbamate BP conjugate (13) wasevaluated.

Compound 13 (an O-thiocarbamate BP Conjugate)

Compound 13 can also be referred to as1-cyclopropyl-7-(4-((4-(2,2-diphosphonoethyl)phenoxy)carbonothioyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicacid. Compound 13 was synthesized as follows. Tetraisopropyl(2-(4-hydroxyphenyl)ethane-1,1-diyl)bis(phosphonate) (0.10 mmol) wasemulsified in water and cooled in an ice bath while stirring vigorously.1,1′-Thiocarbonyldiimidazole (0.12 mmol) was added and allowed to stirfor 1 hour. The ice bath was then removed and stirring continued at roomtemperature for 1 more hour. Ciprofloxacin (0.12 mmol) was then addedand the reaction was stirred overnight at room temperature while coveredwith foil to avoid light. The next day, the white paste was filteredusing a frit funnel and the solids were washed with water and thenether. The solids were collected and purified by silica columnchromatography using a MeOH:CHCl₃ gradient to afford an off white solid.The solid was dissolved in DCM and bromotrimethylsilane (BTMS) (4.00mmol) was added and heated at 35° C. in an oil bath overnight. Solventand BTMS were removed by evaporation and MeOH was added and allowed tostir at room temperature for 30 minutes. Solvent was removed onrotavapor and the product was precipitated in chilled MeOH. Thesuspension was filtered using a frit funnel and washed with additionalMeOH. The solid was collected and excess solvent evaporated to affordthe target compound.

FIG. 24 shows a graph and image demonstrating results from an evaluationof the MIC of an O-thiocarbamate BP conjugate against planktonic S.aureus strain ATCC 6538: negative control=medium+microbes withoutconjugate treatment; positive control=sterile medium without microbes.

FIG. 25 shows a graph demonstrating results from an evaluation of theantimicrobial activity or bacterial load reduction of the thiocarbamateconjugate against biofilms of S. aureus strain ATCC 6538 formed onpolystyrene as the substrate: negative control=microbial dilutionwithout conjugate treatment; positive control=sterile dilution withoutmicrobes.

FIG. 26 shows a graph demonstrating results from an evaluation of theantimicrobial activity of the O-thiocarbamate BP conjugate testedagainst preformed biofilms of S. aureus ATCC 6538 on hydroxyapatite asthe substrate; negative control=microbial dilution without conjugatetreatment. (*p<0.05, Kruskal-Wallis test; triplicate;comparator=control).

FIG. 27 shows a graph demonstrating results from a study usingO-thiocarbamate BP conjugate-treated hydroxyapatite discs evaluating theability to prevent biofilm formation of S. aureus ATCC 6538; negativecontrol=microbial dilution without conjugate treatment. (*p<0.05,Kruskal-Wallis test; triplicate; comparator=control).

FIG. 28 shows a graph demonstrating results from a study usingO-thiocarbamate BP conjugate-treated hydroxyapatite powder evaluatingthe ability to prevent biofilm formation of S. aureus ATCC 6538;negative control=microbial dilution without conjugate treatment.(*p<0.05, Kruskal-Wallis test; triplicate; comparator=control).

Example 11

Described in this example are additional exemplary BP-conjugates andtheir synthesis.

1-cyclopropyl-7-(4-((4-(2,2-diphosphonoethyl)phenoxy)carbonyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (6)

Ciprofloxacin (0.12 mmol) was suspended in water and the pH was adjustedto 8.5 using Na₂CO₃. The suspension was cooled in an ice bath and4-(2,2-bis(diisopropoxyphosphoryl)ethyl)phenyl (4-nitrophenyl) carbonate(0.10 mmol) dissolved in THF was added dropwise. Reaction mixture wasthen removed from ice bath, protected from light and stirred overnightat room temperature. The following day, reaction mixture was dilutedwith water and filtered through a fine glass frit funnel. The retainedsolid was washed with water until no yellow color remained. The solidwas then dissolved and washed from the frit funnel using DCM. Therecovered crude was further purified on a silica column using a MeOH:DCMgradient. Title compound was afforded as a white solid which wasdissolved in DCM and bromotrimethylsilane (BTMS) (4.00 mmol) was addedand heated at 35° C. in an oil bath overnight. Solvent and BTMS wereremoved by evaporation and MeOH was added and allowed to stir at roomtemperature for 30 minutes. Solvent was removed on rotavapor and theproduct was precipitated in chilled MeOH. The suspension was filteredusing a frit funnel and washed with additional MeOH. The solid wascollected and excess solvent removed evaporated to afford the targetcompound.

1-cyclopropyl-6-fluoro-7-(4-((4-(2-hydroxy-2,2-diphosphonoethyl)phenoxy)carbonyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (12)

(1-hydroxy-2-(4-hydroxyphenyl)ethane-1,1-diyl)bis(phosphonic acid) (0.10mmol) was dissolved in water and cooled in an ice bath while stirringvigorously. 1,1′-Carbonyldiimidazole (0.12 mmol) was added and allowedto stir for 1 hour. The ice bath was then removed and stirring continuedat room temperature for 1 more hour. Ciprofloxacin (0.12 mmol) was thenadded and the reaction was stirred overnight at room temperature whilecovered with foil to avoid light. The next day, solvent was removed byevaporation and MeOH was added to precipitate the product. Thesuspension was filtered using a frit funnel and washed with additionalMeOH. The solid was collected and excess solvent evaporated to affordthe target compound.

1-cyclopropyl-7-(4-((4-(2,2-diphosphonoethyl)phenoxy)carbonothioyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (13)

Tetraisopropyl (2-(4-hydroxyphenyl)ethane-1,1-diyl)bis(phosphonate)(0.10 mmol) was emulsified in water and cooled in an ice bath whilestirring vigorously. 1,1′-Thiocarbonyldiimidazole (0.12 mmol) was addedand allowed to stir for 1 hour. The ice bath was then removed andstirring continued at room temperature for 1 more hour. Ciprofloxacin(0.12 mmol) was then added and the reaction was stirred overnight atroom temperature while covered with foil to avoid light. The next day,the white paste was filtered using a frit funnel and the solids werewashed with water and then ether. The solids were collected and purifiedby silica column chromatography using a MeOH:CHCl₃ gradient to afford anoff white solid. The solid was dissolved in DCM and bromotrimethylsilane(BTMS) (4.00 mmol) was added and heated at 35° C. in an oil bathovernight. Solvent and BTMS were removed by evaporation and MeOH wasadded and allowed to stir at room temperature for 30 minutes. Solventwas removed on rotavapor and the product was precipitated in chilledMeOH. The suspension was filtered using a frit funnel and washed withadditional MeOH. The solid was collected and excess solvent evaporatedto afford the target compound.

1-cyclopropyl-6-fluoro-7-(4-((4-(2-hydroxy-2,2-diphosphonoethyl)phenoxy)carbonothioyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (14)

(1-hydroxy-2-(4-hydroxyphenyl)ethane-1,1-diyl)bis(phosphonic acid) (0.10mmol) was dissolved in water and cooled in an ice bath while stirringvigorously. 1,1′-Thiocarbonyldiimidazole (0.12 mmol) was added andallowed to stir for 1 hour. The ice bath was then removed and stirringcontinued at room temperature for 1 more hour. Ciprofloxacin (0.12 mmol)was then added and the reaction was stirred overnight at roomtemperature while covered with foil to avoid light. The next day,solvent was removed by evaporation and MeOH was added to precipitate theproduct. The suspension was filtered using a frit funnel and washed withadditional MeOH. The solid was collected and excess solvent evaporatedto afford the target compound.

1-cyclopropyl-7-(4-(((4-(2,2-diphosphonoethyl)phenyl)thio)carbonyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid(15)

In a microwave vial, compound 13 was suspended on NMP and heated at 290°C. in a microwave reactor for 20 minutes. The suspension was filteredand washed with MeOH to afford the target compound.

1-cyclopropyl-6-fluoro-7-(4-(((4-(2-hydroxy-2,2-diphosphonoethyl)phenyl)thio)carbonyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (16)

In a microwave vial, compound 14 was suspended on NMP and heated at 290°C. in a microwave reactor for 20 minutes. The suspension was filteredand washed with MeOH to afford the target compound.

1-cyclopropyl-7-((4aR,7aR)-1-((4-(2,2-diphosphonoethyl)phenoxy)carbonyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (17)

Compound 17 was synthesized according to the procedure described forcompound 6, replacing ciprofloxacin with moxifloxacin.

1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-((4-(2-hydroxy-2,2-diphosphonoethyl)phenoxy)carbonyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (18)

Compound 18 was synthesized according to the procedure described forcompound 12, replacing ciprofloxacin with moxifloxacin.

1-cyclopropyl-7-((4aR,7aR)-1-((4-(2,2-diphosphonoethyl)phenoxy)carbonothioyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (19)

Compound 19 was synthesized according to the procedure described forcompound 13, replacing ciprofloxacin with moxifloxacin.

1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-((4-(2-hydroxy-2,2-diphosphonoethyl)phenoxy)carbonothioyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (20)

Compound 20 was synthesized according to the procedure described forcompound 14, replacing ciprofloxacin with moxifloxacin.

1-cyclopropyl-7-((4aR,7aR)-1-(((4-(2,2-diphosphonoethyl)phenyl)thio)carbonyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (21)

In a microwave vial, compound 19 was suspended on NMP and heated at 290°C. for 20 minutes. The suspension was filtered and washed with MeOH toafford the target compound.

1-cyclopropyl-6-fluoro-7-((4aR,7aR)-1-(((4-(2-hydroxy-2,2-diphosphonoethyl)phenyl)thio)carbonyl)octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (22)

In a microwave vial, compound 20 was suspended on NMP and heated at 290°C. for 20 minutes. The suspension was filtered and washed with MeOH toafford the target compound.

1-cyclopropyl-7-(4-((4-(2,2-diphosphonoethyl)phenoxy)carbonyl)-3-methylpiperazin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (23)

Compound 23 was synthesized according to the procedure described forcompound 6, replacing ciprofloxacin with gatifloxacin.

1-cyclopropyl-6-fluoro-7-(4-((4-(2-hydroxy-2,2-diphosphonoethyl)phenoxy)carbonyl)-3-methylpiperazin-1-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (24)

Compound 24 was synthesized according to the procedure described forcompound 12, replacing ciprofloxacin with gatifloxacin.

1-cyclopropyl-7-(4-((4-(2,2-diphosphonoethyl)phenoxy)carbonothioyl)-3-methylpiperazin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (25)

Compound 25 was synthesized according to the procedure described forcompound 13, replacing ciprofloxacin with gatifloxacin.

1-cyclopropyl-6-fluoro-7-(4-((4-(2-hydroxy-2,2-diphosphonoethyl)phenoxy)carbonothioyl)-3-methylpiperazin-1-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (26)

Compound 26 was synthesized according to the procedure described forcompound 14, replacing ciprofloxacin with gatifloxacin.

1-cyclopropyl-7-(4-(((4-(2,2-diphosphonoethyl)phenyl)thio)carbonyl)-3-methylpiperazin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (27)

In a microwave vial, compound 25 was suspended on NMP and heated at 290°C. for 20 minutes. The suspension was filtered and washed with MeOH toafford the target compound.

1-cyclopropyl-6-fluoro-7-(4-(((4-(2-hydroxy-2,2-diphosphonoethyl)phenyl)thio)carbonyl)-3-methylpiperazin-1-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (28)

In a microwave vial, compound 26 was suspended on NMP and heated at 290°C. for 20 minutes. The suspension was filtered and washed with MeOH toafford the target compound.

7-(4-((4-(2,2-diphosphonoethyl)phenoxy)carbonyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (29)

Compound 29 was synthesized according to the procedure described forcompound 6, replacing ciprofloxacin with norfloxacin.

1-ethyl-6-fluoro-7-(4-((4-(2-hydroxy-2,2-diphosphonoethyl)phenoxy)carbonyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (30)

Compound 30 was synthesized according to the procedure described forcompound 12, replacing ciprofloxacin with norfloxacin.

7-(4-((4-(2,2-diphosphonoethyl)phenoxy)carbonothioyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (31)

Compound 31 was synthesized according to the procedure described forcompound 13, replacing ciprofloxacin with norfloxacin.

1-ethyl-6-fluoro-7-(4-((4-(2-hydroxy-2,2-diphosphonoethyl)phenoxy)carbonothioyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (32)

Compound 32 was synthesized according to the procedure described forcompound 14, replacing ciprofloxacin with norfloxacin.

7-(4-(((4-(2,2-diphosphonoethyl)phenyl)thio)carbonyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (33)

In a microwave vial, compound 31 was suspended on NMP and heated at 290°C. for 20 minutes. The suspension was filtered and washed with MeOH toafford the target compound.

1-ethyl-6-fluoro-7-(4-(((4-(2-hydroxy-2,2-diphosphonoethyl)phenyl)thio)carbonyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (34)

In a microwave vial, compound 32 was suspended on NMP and heated at 290°C. for 20 minutes. The suspension was filtered and washed with MeOH toafford the target compound.

1-cyclopropyl-7-(4-(((4-(2,2-diphosphonoethyl)phenyl)thio)carbonothioyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (35)

1-cyclopropyl-6-fluoro-7-(4-(((4-(2-hydroxy-2,2-diphosphonoethyl)phenyl)thio)carbonothioyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (36) Example 12 1. Dimethyl acetylphosphonate (XV-PC-055) (37)

Trimethyl phosphite (2.36 mL, 20 mmol) was added to ice-cold acetylchloride (1.44 mL, 20.2 mmol) under N₂ over a period of 20 mins. Thecolorless solution was warmed to room temperature, stirred for 30 mins,and concentrated under vacuum to afford 2.89 g (94%) product ascolorless oil which was used in next reaction as is. ¹HNMR (300 MHz,CDCl3): δ 3.84 (d, J=12 Hz, 6H), 2.46 (d, J=5.4 Hz, 3H). ³¹PNMR (121MHz, CDCl₃): δ −1.10.

2. Tetramethyl(1-hydroxyethylidene)-bisphosphonate (XV-PC-057) (38)

Dimethyl acetylphosphate (37) (2.2 g, 14.44 mmol) was added dropwiselyto an ice-cold solution of dimethyl phosphite (1.63 mL, 15.91 mmol) anddibutylamine (0.767 mL, 1.44 mmol) in dry ether (30 mL) under N₂. Theice bath was removed, and the mixture was stirred at room temperaturefor 3 h. The resulting precipitate was filtered, washed with ether, anddried under vacuum overnight to afford 3.24 g (85%) of product as whitesolid. ¹HNMR (300 MHz, CDCl₃): δ 3.94-3.82 (m, 12H), 3.44 (t, J=8.4 Hz,1H), 1.68 (t, J=16.2 Hz, 3H). ³¹PNMR (121 MHz, CDCl₃): δ 22.21. MS-ESI:263.1 [M+H]+.

3. Tetramethyl(1-{[(4-nitrophenoxy)carbonyl]oxy}ethane-1,1-diyl)bis(phosphonate)(XV-PC-099) (39)

p-nitrophenyl chloroformate (768 mg, 3.81 mmol) was added to an ice-coldsolution of DMAP (466 mg, 3.81 mmol) in DCM (20 mL) under N₂. Afterstirring for 10 mins, thetetramethyl(1-hydroxyethylidene)-bisphosphonate (1 g, 3.81 mmol) wasadded in one portion. The ice-bath was removed, and the mixture wasstirred at room temperature for 3 h. Next, the reaction mixture wasextracted with 20 mL each of cold aqueous 0.1 N HCl (2×), water, brine,dried over MgSO₄, and concentrated. The crude mixture was separated bycolumn chromatography using EtOAc/MeOH (1-3%) to afford 1.16 g (71%)light-yellow oil. ¹HNMR (300 MHz, CDCl3): δ 8.27 (d, J=9 Hz, 2H), 7.40(d, J=9 Hz, 2H), 3.97-3.87 (m, 12H), 2.02 (t, J=15.6 Hz, 3H). ³¹PNMR(121 MHz, CDCl₃): δ 17.98. MS-ESI: 445.3 [M+NH4]+.

4: Ciprofloxacin carbamoyl etidronate tetramethyl Ester (XV-PC-101) (40)

To a solution of NaHCO₃ (239.5 mg, 2.85 mmol) in H₂O (20 mL) was addedciprofloxacin (899.6 mg, 2.71 mmol) and the suspension was cooled inice-bath. Next, the tetramethyl(1-{[(4-nitrophenoxy)carbonyl]oxy}ethane-1,1-diyl)bis(phosphonate)dissolved in THF (20 mL) was added dropwisely over a period of 20 mins.The yellow suspension was stirred overnight (14 h) at room temperature.The reaction mixture was concentrated, and the crude mixture wasseparated by column chromatography using DCM/MeOH (1-5%) to provide 832mg (49%) of light-yellow solid. ¹HNMR (300 MHz, CDCl₃): δ 8.77 (s, 1H),8.03 (d, J=12.6 Hz, 1H), 7.36 (d, J=6.9 Hz, 1H) 3.98-3.80 (m, 12H),3.79-3.68 (br s, 4H), 3.58-3.50 (m, 1H), 3.30 (t, J=9.6 Hz, 4H), 1.95(t, J=15.6 Hz, 3H), 1.40 (q, J=6.8 Hz, 2H), 1.23-1.16 (m, 2H). ³¹PNMR(121 MHz, CDCl3): δ 20.19. MS-ESI: 620.3 [M+H]+.

5. Etidronate-carbamate-Ciprofloxacin (XV-PC-105) (41)

A mixture of tetramethyl etidronate-carbamate-ciprofloxacin (775 mg,1.25 mmol) and bromotrimethylsilane (1.53 g, 10 mmol) in ACN (28 mL) wasstirred for 2 h. The volatiles were evaporated under vacuum and MeOH (28mL) was added to the residue. After stirring for 30 mins the resultingsuspension was filtered, washed with MeOH (10 mL×2), and dried undervacuum overnight to afford 662 mg (93%) off-white solid. ¹H NMR (300MHz, 20% CD₃CN in DMSO-d6) δ 8.66 (s, 1H), 7.92 (d, J=13.2 Hz, 1H), 7.57(d, J=7.4 Hz, 1H), 3.78 (p, J=3.1 Hz, 1H), 3.64 (br d, J=32.1 Hz, 4H),3.32 (br s, 4H), 1.82 (t, J=15.1 Hz, 3H), 1.32 (d, J=6.5 Hz, 2H), 1.16(s, 2H). ³¹PNMR (121 MHz, 20% CD3CN in DMSO-d6): δ 15.57. MS-ESI: 564.2[M+H]+.

6. Moxifloxacin carbamoyl etidronate tetramethyl Ester (XVI-PC-029) (42)

Moxifloxacin HCl was added to a solution of Na2CO3 in H2O (20 mL) andthe solution was cooled in ice bath. Next, the tetramethyl(1-{[(4-nitrophenoxy)carbonyl]oxy}ethane-1,1-diyl)bis(phosphonate)dissolved in THF (20 mL) was added dropwisely over 30 min. The ice bathwas removed, the flask was covered with aluminum foil, and the reactionwas stirred for 20 h at room temperature. Next, the reaction mixture wasconcentrated, and the crude purified by column chromatography usingDCM/MeOH (1-5%) to afford 624 mg (29%) of product as off-white foam. ¹HNMR (300 MHz, Chloroform-d) δ 8.78 (s, 1H), 7.81 (d, J=13.8 Hz, 1H),4.82 (br s, 1H), 4.16-4.04 (m, 2H), 4.02-3.92 (m, 2H), 3.92-3.80 (m,12H), 3.56 (s, 3H), 3.48 (t, J=10.5 Hz, 1H), 3.24 (d, J=10.5 Hz, 1H),3.00 (br s, 1H), 2.40-2.24 (m, 1H), 1.94 (t, J=15.9 Hz, 3H), 1.87-1.74(m, 2H), 1.60-1.44 (m, 2H), 1.35-1.21 (m, 1H), 1.17-1.01 (m, 2H),0.88-0.75 (m, 1H). ³¹PNMR (121 MHz, CDCl3): δ 20.36. MS-ESI: 690.4[M+H]+

7. Etidronate-carbamate-Moxifloxacin (XVI-PC-033) (43)

A mixture of tetramethyl etidronate-carbamate-moxifloxacin tetramethylester (764 mg, 1.10 mmol) and bromotrimethylsilane (1.35 g, 8.86 mmol)in ACN (25 mL) was stirred for 2 h. The volatiles were evaporated undervacuum and MeOH (25 mL) was added to the residue. After stirring for 30mins, the solvent was evaporated, and the residue was triturated withminimum volume of DCM for 30 mins. The solid was filtered, and driedunder high vacuum to afford 757 mg of product (quantitative yield). ¹HNMR (300 MHz, Methanol-d4) δ 8.98 (s, 1H), 7.79 (d, J=14.5 Hz, 1H),4.39-4.25 (m, 1H), 4.24-4.07 (m, 2H), 4.01 (t, J=10.3 Hz, 1H), 3.65 (s,3H), 3.61-3.51 (m, 2H), 3.41 (d, J=10.7 Hz, 1H), 3.04 (br s, 1H),2.43-2.27 (m, 1H), 1.90 (t, J=15.2 Hz, 3H), 1.83-1.71 (m, 2H), 1.55 (q,J=10.8 Hz, 2H), 1.43-1.32 (m, 1H), 1.30-1.18 (m, 1H), 1.17-1.03 (m, 1H),1.01-0.83 (m, 1H). ³¹PNMR (121 MHz, Methanol-d4): δ 16.60. MS-ESI: 634.2[M+H]+.

Example 13

The following is a general structure of BP-quinolone as can be describedin one or more aspects herein.

conjugates between alpha-OH containing BP and fluoroquinolone

Example 14

The following are non-limiting examples of BP-quinolone conjugates asdescribed in one or more aspects herein.

We claim:
 1. The compound of claim 4, wherein the compound is accordingto Formula (6)


2. The pharmaceutical formulation of claim 12, wherein the compound isaccording to Formula (6)


3. A method of treating or preventing a bone infection in a subject inneed thereof, the method comprising: administering an amount of thecompound of claim 1, or a pharmaceutical formulation comprising thecompound of claim 1 and a pharmaceutically acceptable carrier, to thesubject in need thereof.
 4. A compound comprising: a bisphosphonate; anda quinolone compound; wherein the quinolone compound is releasablycoupled to the bisphosphonate via a linker, wherein the linker is acarbamate linker.
 5. The compound of claim 4, wherein the bisphosphonateis selected from the group consisting of: hydroxyl phenyl alkyl or arylbisphosphonates, hydroxyl phenyl (or aryl) alkyl hydroxylbisphosphonates, amino phenyl(or aryl) alkyl bisphosphonates, aminophenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkylbisphosphonates, hydroxyl alkyl hydroxyl bisphosphonates, hydroxyl alkylphenyl(or aryl) alkyl bisphosphonates, hydroxyl phenyl(or aryl) alkylhydroxyl bisphosphonates, amino phenyl(or aryl) alkyl bisphosphonates,amino phenyl(or aryl) alkyl hydroxyl bisphosphonates, hydroxyl alkylbisphosphonates, hydroxyl alkyl hydroxyl bisphosphonates, hydroxypyridylalkyl bisphosphonates, pyridyl alkyl bisphosphonates, hydroxyl imadazoylalkyl bisphosphonates, imidazoyl alkyl bisphosphonates, etidronate,pamidronate, neridronate, olpadronate, alendronate, ibandronate,risedronate, zoledronate, minodronate and combinations thereof, whereinall the compounds are optionally further substituted or areunsubstituted.
 6. The compound of claim 4, wherein the quinolonecompound is selected from the group consisting of: alatrofloxacin,amifloxacin, balofloxacin, besifloxacin, cadazolid, ciprofloxacin,clinafloxacin, danofloxacin, delafloxacin, difloxacin, enoxacin,enrofloxacin, finafloxacin, flerofloxacin, flumequine, gatifloxacin,gemifloxacin, grepafloxacin, ibafloxacin, JNJ-Q2, levofloxacin,lomefloxacin, marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin,ofloxacin, orbifloxacin, pazufloxacin, pefloxacin, pradofloxacin,prulifloxacin, rufloxacin, sarafloxacin, sitafloxacin, sparfloxacin,temafloxacin, tosufloxacin, trvafloxacin, zabofloxacin, nemonoxacin andcombinations thereof.
 7. The compound of claim 4, wherein the quinolonecompound has a structure according to Formula A,

where R¹ is one or more substituents selected from the group consistingof: an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substitutedalkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy,alkylthio, substituted alkylthio, phenylthio, substituted phenylthio,arylthio, substituted arylthio, cyano, isocyano, substituted isocyano,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, sulfonyl, substitutedsulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups, where R² is selected from thegroup consisting of: an alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl,alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy,substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano,substituted isocyano, carbonyl, substituted carbonyl, carboxyl,substituted carboxyl, amino, substituted amino, amido, substitutedamido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl,substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups, where R³ is selected from the group consisting of:an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substitutedalkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy,alkylthio, substituted alkylthio, phenylthio, substituted phenylthio,arylthio, substituted arylthio, cyano, isocyano, substituted isocyano,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, sulfonyl, substitutedsulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups, and where R⁴ is selected from thegroup consisting of: an alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl,alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy,substituted aroxy, alkylthio, substituted alkylthio, phenylthio,substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano,substituted isocyano, carbonyl, substituted carbonyl, carboxyl,substituted carboxyl, amino, substituted amino, amido, substitutedamido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl,substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl,substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀ cyclic,heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups.
 8. The compound of claim 4, wherein the linker is anaryl carbamate, an aryl thiocarbamate, an O-thioarylcarbamate, anS-thioarylcarbamate, a thiocarbamate, an O-thiocarbamate, anS-thiocarbamate, a phenyl carbamate, a phosphonyl carbamate, or an arylor phosphonyl substituted carbamate linker.
 9. The compound of claim 8,wherein the linker is attached to the R¹ group of Formula A.
 10. Thecompound of claim 4, wherein the bisphosphonate isethylidenebisphosphonate and the alpha position of theethylidenebisphosphonate is substituted by hydroxy, fluoro, chloro,bromo or iodo.
 11. The compound of claim 4, wherein the compound has aformula according to Formula (12):

a formula according to Formula (13)

a formula according to Formula (15)

a formula according to Formula (41)

or a formula according to Formula (43)


12. A pharmaceutical formulation comprising: an amount of a compound asset forth in claim 4; and a pharmaceutically acceptable carrier.
 13. Thepharmaceutical formulation of claim 12, wherein the amount of thecompound is an amount effective to kill or inhibit bacteria growth, totreat or prevent bone diseases with abnormal bone resorption, to treator prevent bone infections, or to treat or prevent osteoporosis,osteomyelitis, osteonecrosis, peri-implantitis, and periodontitis
 14. Amethod of treating or preventing osteomyelitis in a subject in needthereof, the method comprising: administering an amount of a compoundclaim 4, or a pharmaceutical formulation comprising a compound of claim4 and a pharmaceutically acceptable carrier, to the subject in needthereof.
 15. A method of treating or preventing peri-implantitis orperiodontitis in a subject in need thereof, the method comprisingadministering an amount of a compound as in claim 4, or a pharmaceuticalformulation comprising a compound of claim 4 and a pharmaceuticallyacceptable carrier, to the subject in need thereof.
 16. A method oftreating or preventing diabetic foot in a subject in need thereof, themethod comprising administering an amount of a compound as in claim 4,or a pharmaceutical formulation comprising a compound of claim 4 and apharmaceutically acceptable carrier, to the subject in need thereof. 17.A bone graft composition comprising: a bone graft material and acompound as in claim 4, or a pharmaceutical formulation comprising acompound of claim 4 and a pharmaceutically acceptable carrier, whereinthe compound or pharmaceutical formulation is attached to, integratedwith, chemisorbed to, or mixed with the bone graft material, or whereinthe bone graft material is autograft bone material, allograft bonematerial, xenograft bone material, a synthetic bone graft material, orany combination thereof.
 18. A method comprising: implanting the bonegraft composition of claim 17 into a subject in need thereof.
 19. Amethod of treating or preventing biofilm infection at an osseous orimplant surgical site, or at a surgical site where bone grafting isperformed, where the method comprises: administering a compound claim 4,or a pharmaceutical formulation comprising a compound of claim 4 and apharmaceutically acceptable carrier, to a subject in need thereof.
 20. Amethod of treating or preventing biofilm infection at an osseous orimplant surgical site, or at a surgical site where bone grafting isperformed, where the method comprises: implanting a bone graftcomposition as in claim 17 to a subject in need thereof.